Integrase Defective Lentiviral Vector Promoter Impacts Transgene Expression in Target Cells and Magnitude of Vector-Induced Immune Responses.

Integrase defective lentiviral vectors (IDLVs) are a promising vaccine delivery platform given their ability to induce high magnitude and durable antigen-specific immune responses. IDLVs based on the simian immunodeficiency virus (SIV) are significantly more efficient at transducing human and simian dendritic cells (DCs) compared to HIV-based vectors, resulting in a higher expansion of antigen-specific CD8+ T cells. Additionally, IDLV persistence and continuous antigen expression in muscle cells at the injection site contributes to the durability of the vaccine-induced immune responses. Here, to further optimize transgene expression levels in both DCs and muscle cells, we generated ten novel lentiviral vectors (LVs) expressing green fluorescent protein (GFP) under different hybrid promoters. Our data show that three of the tested hybrid promoters resulted in the highest transgene expression levels in mouse DCs, monkey DCs and monkey muscle cells. We then used the three LVs with the highest in vitro transgene expression levels to immunize BALB/c mice and observed high magnitude T cell responses at 3 months post-prime. Our study demonstrates that the choice of the vector promoter influences antigen expression levels in target cells and the ensuing magnitude of T cell responses in vivo.

Dam-Infant Rhesus Macaque Pairs to Dissect Age-Dependent Responses to SARS-CoV-2 Infection.

The global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated coronavirus disease (COVID-19) has led to a pandemic of unprecedented scale. An intriguing feature of the infection is the minimal disease in most children, a demographic at higher risk for other respiratory viral diseases. To investigate age-dependent effects of SARS-CoV-2 pathogenesis, we inoculated two rhesus macaque monkey dam-infant pairs with SARS-CoV-2 and conducted virological and transcriptomic analyses of the respiratory tract and evaluated systemic cytokine and Ab responses. Viral RNA levels in all sampled mucosal secretions were comparable across dam-infant pairs in the respiratory tract. Despite comparable viral loads, adult macaques showed higher IL-6 in serum at day 1 postinfection whereas CXCL10 was induced in all animals. Both groups mounted neutralizing Ab responses, with infants showing a more rapid induction at day 7. Transcriptome analysis of tracheal airway cells isolated at day 14 postinfection revealed significant upregulation of multiple IFN-stimulated genes in infants compared with adults. In contrast, a profibrotic transcriptomic signature with genes associated with cilia structure and function, extracellular matrix composition and metabolism, coagulation, angiogenesis, and hypoxia was induced in adults compared with infants. Our study in rhesus macaque monkey dam-infant pairs suggests age-dependent differential airway responses to SARS-CoV-2 infection and describes a model that can be used to investigate SARS-CoV-2 pathogenesis between infants and adults.

The use of viral vectors in vaccine development.

Vaccines represent the single most cost-efficient and equitable way to combat and eradicate infectious diseases. While traditional licensed vaccines consist of either inactivated/attenuated versions of the entire pathogen or subunits of it, most novel experimental vaccines against emerging infectious diseases employ nucleic acids to produce the antigen of interest directly in vivo. These include DNA plasmid vaccines, mRNA vaccines, and recombinant viral vectors. The advantages of using nucleic acid vaccines include their ability to induce durable immune responses, high vaccine stability, and ease of large-scale manufacturing. In this review, we present an overview of pre-clinical and clinical data on recombinant viral vector vaccines and discuss the advantages and limitations of the different viral vector platforms.

Establishment, Persistence, and Reactivation of Latent HIV-1 Infection in Renal Epithelial Cells.

HIV-1 persistence in different cell types presents the main obstacle to an HIV-1 cure. We have previously shown that the renal epithelium is a site of HIV-1 infection and that the kidney represents a separate viral compartment from blood. Whether renal cells can harbor latent virus that can be reactivated upon treatment with latency reversing agents (LRAs) is unknown. To address this question, we developed an in vitro HIV-1 latency model in renal tubule epithelial (RTE) cells using a dual color HIV-1 reporter virus, R7/E-/GFP/EF1a-mCherry (R7GEmC), and evaluated the effect of LRAs, both as single agents and in combination, on viral reactivation. Our data show that HIV-1 can establish latency in RTE cells early postinfection. While the pool of latently infected cells expanded overtime, the percentage of productively infected cells declined. Following LRA treatment only a small fraction of latently infected cells, both T cells and RTE cells, could be reactivated, and the drug combinations more effective in reactivating HIV transcription in RTE cells differed from those more active in T cells. Our study demonstrates that HIV can establish latency in RTE cells and that current LRAs are only marginally effective in inducing HIV-1 reactivation. This suggests that further study of LRA dynamics in non-T cells may be warranted to assess the suitability of LRAs as a sterilizing cure strategy. IMPORTANCE Anti-retroviral therapy (ART) has dramatically reduced HIV-related morbidity and mortality. Despite this success, a number of challenges remain, including the long-term persistence of multiple, clinically latent viral reservoirs capable of reactivation in the absence of ART. As efforts proceed toward HIV eradication or functional cure, further understanding of the dynamics of HIV-1 replication, establishment of latency and mechanisms of reactivation in reservoirs harboring the virus throughout the body is necessary. HIV-1 can infect renal epithelial cells and the expression of viral genes in those cells contributes to the development of HIV associated nephropathy (HIVAN) in untreated individuals. The significance of our work is in developing the first model of HIV-1 latency in renal epithelial cells. This model enhances our understanding of HIV-1 latency and persistence in the kidney and can be used to screen candidate latency reversing agents.

SARS-CoV-2 Employ BSG/CD147 and ACE2 Receptors to Directly Infect Human Induced Pluripotent Stem Cell-Derived Kidney Podocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the Coronavirus disease 2019 (COVID-19), which has resulted in over 5.9 million deaths worldwide. While cells in the respiratory system are the initial target of SARS-CoV-2, there is mounting evidence that COVID-19 is a multi-organ disease. Still, the direct affinity of SARS-CoV-2 for cells in other organs such as the kidneys, which are often targeted in severe COVID-19, remains poorly understood. We employed a human induced pluripotent stem (iPS) cell-derived model to investigate the affinity of SARS-CoV-2 for kidney glomerular podocytes, and examined the expression of host factors for binding and processing of the virus. We studied cellular uptake of the live SARS-CoV-2 virus as well as a pseudotyped virus. Infection of podocytes with live SARS-CoV-2 or spike-pseudotyped lentiviral particles revealed cellular uptake even at low multiplicity of infection (MOI) of 0.01. We found that direct infection of human iPS cell-derived podocytes by SARS-CoV-2 virus can cause cell death and podocyte foot process retraction, a hallmark of podocytopathies and progressive glomerular diseases including collapsing glomerulopathy observed in patients with severe COVID-19 disease. We identified BSG/CD147 and ACE2 receptors as key mediators of spike binding activity in human iPS cell-derived podocytes. These results show that SARS-CoV-2 can infect kidney glomerular podocytes in vitro via multiple binding interactions and partners, which may underlie the high affinity of SARS-CoV-2 for kidney tissues. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.

E-cigarette and food flavoring diacetyl alters airway cell morphology, inflammatory and antiviral response, and susceptibility to SARS-CoV-2.

Diacetyl (DA) is an α-diketone that is used to flavor microwave popcorn, coffee, and e-cigarettes. Occupational exposure to high levels of DA causes impaired lung function and obstructive airway disease. Additionally, lower levels of DA exposure dampen host defenses in vitro. Understanding DA's impact on lung epithelium is important for delineating exposure risk on lung health. In this study, we assessed the impact of DA on normal human bronchial epithelial cell (NHBEC) morphology, transcriptional profiles, and susceptibility to SARS-CoV-2 infection. Transcriptomic analysis demonstrated cilia dysregulation, an increase in hypoxia and sterile inflammation associated pathways, and decreased expression of interferon-stimulated genes after DA exposure. Additionally, DA exposure resulted in cilia loss and increased hyaluronan production. After SARS-CoV-2 infection, both genomic and subgenomic SARS-CoV-2 RNA were increased in DA vapor- compared to vehicle-exposed NHBECs. This work suggests that transcriptomic and physiologic changes induced by DA vapor exposure damage cilia and increase host susceptibility to SARS-CoV-2.

BSG/CD147 and ACE2 receptors facilitate SARS-CoV-2 infection of human iPS cell-derived kidney podocytes.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the Coronavirus disease 2019 (COVID-19), which was declared a pandemic by the World Health Organization (WHO) in March 2020. The disease has caused more than 5.1 million deaths worldwide. While cells in the respiratory system are frequently the initial target for SARS-CoV-2, clinical studies suggest that COVID-19 can become a multi-organ disease in the most severe cases. Still, the direct affinity of SARS-CoV-2 for cells in other organs such as the kidneys, which are often affected in severe COVID-19, remains poorly understood. METHOD: In this study, we employed a human induced pluripotent stem (iPS) cell-derived model to investigate the affinity of SARS-CoV-2 for kidney glomerular podocytes. We studied uptake of the live SARS-CoV-2 virus as well as pseudotyped viral particles by human iPS cell derived podocytes using qPCR, western blot, and immunofluorescence. Global gene expression and qPCR analyses revealed that human iPS cell-derived podocytes express many host factor genes (including ACE2, BSG/CD147, PLS3, ACTR3, DOCK7, TMPRSS2, CTSL CD209, and CD33) associated with SARS-CoV-2 binding and viral processing. RESULT: Infection of podocytes with live SARS-CoV-2 or spike-pseudotyped lentiviral particles revealed viral uptake by the cells at low Multiplicity of Infection (MOI of 0.01) as confirmed by RNA quantification and immunofluorescence studies. Our results also indicate that direct infection of human iPS cell-derived podocytes by SARS-CoV-2 virus can cause cell death and podocyte foot process retraction, a hallmark of podocytopathies and progressive glomerular diseases including collapsing glomerulopathy observed in patients with severe COVID-19 disease. Additionally, antibody blocking experiments identified BSG/CD147 and ACE2 receptors as key mediators of spike binding activity in human iPS cell-derived podocytes. CONCLUSION: These results show that SARS-CoV-2 can infect kidney glomerular podocytes in vitro . These results also show that the uptake of SARS-CoV-2 by kidney podocytes occurs via multiple binding interactions and partners, which may underlie the high affinity of SARS-CoV-2 for kidney tissues. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism. SIGNIFICANT STATEMENT: Many patients with COVID19 disease exhibit multiorgan complications, suggesting that SARS-CoV-2 infection can extend beyond the respiratory system. Acute kidney injury is a common COVID-19 complication contributing to increased morbidity and mortality. Still, SARS-Cov-2 affinity for specialized kidney cells remain less clear. By leveraging our protocol for stem cell differentiation, we show that SARS-CoV-2 can directly infect kidney glomerular podocytes by using multiple Spike-binding proteins including ACE2 and BSG/CD147. Our results also indicate that infection by SARS-CoV-2 virus can cause podocyte cell death and foot process effacement, a hallmark of podocytopathies including collapsing glomerulopathy observed in patients with severe COVID-19 disease. This stem cell-derived model is potentially useful for kidney-specific antiviral drug screening and mechanistic studies of COVID-19 organotropism.