Synergistic effect of two human-like monoclonal antibodies confers protection against orthopoxvirus infection.

The eradication of smallpox was officially declared by the WHO in 1980, leading to discontinuation of the vaccination campaign against the virus. Consequently, immunity against smallpox and related orthopoxviruses like Monkeypox virus gradually declines, highlighting the need for efficient countermeasures not only for the prevention, but also for the treatment of already exposed individuals. We have recently developed human-like monoclonal antibodies (mAbs) from vaccinia virus-immunized non-human primates. Two mAbs, MV33 and EV42, targeting the two infectious forms of the virus, were selected for in vivo evaluation, based on their in vitro neutralization potency. A single dose of either MV33 or EV42 administered three days post-infection (dpi) to BALB/c female mice provides full protection against lethal ectromelia virus challenge. Importantly, a combination of both mAbs confers full protection even when provided five dpi. Whole-body bioimaging and viral load analysis reveal that combination of the two mAbs allows for faster and more efficient clearance of the virus from target organs compared to either MV33 or EV42 separately. The combined mAbs treatment further confers post-exposure protection against the currently circulating Monkeypox virus in Cast/EiJ female mice, highlighting their therapeutic potential against other orthopoxviruses.

Open science discovery of potent noncovalent SARS-CoV-2 main protease inhibitors.

We report the results of the COVID Moonshot, a fully open-science, crowdsourced, and structure-enabled drug discovery campaign targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease. We discovered a noncovalent, nonpeptidic inhibitor scaffold with lead-like properties that is differentiated from current main protease inhibitors. Our approach leveraged crowdsourcing, machine learning, exascale molecular simulations, and high-throughput structural biology and chemistry. We generated a detailed map of the structural plasticity of the SARS-CoV-2 main protease, extensive structure-activity relationships for multiple chemotypes, and a wealth of biochemical activity data. All compound designs (>18,000 designs), crystallographic data (>490 ligand-bound x-ray structures), assay data (>10,000 measurements), and synthesized molecules (>2400 compounds) for this campaign were shared rapidly and openly, creating a rich, open, and intellectual property-free knowledge base for future anticoronavirus drug discovery.

Identification of T-Cell Epitopes Using a Combined In-Silico and Experimental Approach in a Mouse Model for SARS-CoV-2.

Following viral infection, T-cells are crucial for an effective immune response to intracellular pathogens, including respiratory viruses. During the COVID-19 pandemic, diverse assays were required in pre-clinical trials to evaluate the immune response following vaccination against SARS-CoV-2 and assess the response following exposure to the virus. To assess the nature and potency of the cellular response to infection or vaccination, a reliable and specific activity assay was needed. A cellular activity assay based on the presentation of short peptides (epitopes) allows the identification of T cell epitopes displayed on different alleles of the MHC, shedding light on the strength of the immune response towards antigens and aiding in antigen design for vaccination. In this report, we describe two approaches for scanning T cell epitopes on the surface glycoprotein of the SARS-CoV-2 (spike), which is utilized for attachment and entry and serves as an antigen in many vaccine candidates. We demonstrate that epitope scanning is feasible using peptide libraries or computational scanning combined with a cellular activity assay. Our scans identified four CD8 T cell epitopes, including one novel undescribed epitope. These epitopes enabled us to establish a reliable T-cell response assay, which was examined and used in various experimental mouse models for SARS-CoV-2 infection and vaccination. These approaches could potentially aid in future antigen design for vaccination and establish cellular activity assays against uncharacterized antigens of emerging pathogens.

Generation of recombinant mAbs to vaccinia virus displaying high affinity and potent neutralization.

Members of the Orthopoxvirus genus can cause severe infections in humans. Global vaccination against smallpox, caused by the variola virus, resulted in the eradication of the disease in 1980. Shortly thereafter, vaccination was discontinued, and as a result, a large proportion of the current population is not protected against orthopoxviruses. The concerns that the variola virus or other engineered forms of poxviruses may re-emerge as bioweapons and the sporadic outbreaks of zoonotic members of the family, such as Mpox, which are becoming more frequent and prevalent, also emphasize the need for an effective treatment against orthopoxviruses. To date, the most effective way to prevent or control an orthopoxvirus outbreak is through vaccination. However, the traditional vaccinia-based vaccine may cause severe side effects. Vaccinia immune globulin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of vaccine adverse reactions and was also used occasionally for the treatment of severe orthopoxvirus infections. However, this treatment carries many disadvantages and is also in short supply. Thus, a recombinant alternative is highly needed. In this study, two non-human primates were immunized with live vaccinia virus, producing a robust and diverse antibody response. A phage-display library was constructed based on the animal's lymphatic organs, and a panel of neutralizing monoclonal antibodies (mAbs), recognizing diverse proteins of the vaccinia virus, was selected and characterized. These antibodies recognized both mature virion and enveloped virion forms of the virus and exhibited high affinity and potent in vitro neutralization capabilities. Furthermore, these monoclonal antibodies were able to neutralize Mpox 2018 and 2022 strains, suggesting a potential for cross-species protection. We suggest that a combination of these mAbs has the potential to serve as recombinant therapy both for vaccinia vaccine adverse reactions and for orthopoxvirus infections. IMPORTANCE In this manuscript, we report the isolation and characterization of several recombinant neutralizing monoclonal antibodies (mAbs) identified by screening a phage-display library constructed from lymphatic cells collected from immunized non-human primates. The antibodies target several different antigens of the vaccinia virus, covering both mature virion and extracellular enveloped virion forms of the virus. We document strong evidence indicating that they exhibit excellent affinity to their respective antigens and, most importantly, optimal in vitro neutralization of the virus, which exceeded that of vaccinia immune globulin. Furthermore, we present the ability of these novel isolated mAbs (as well as the sera collected from vaccinia-immunized animals) to neutralize two Mpox strains from the 2018 to 2022 outbreaks. We believe that these antibodies have the potential to be used for the treatment of vaccinia vaccine adverse reactions, for other orthopoxvirus infections, and in cases of unexpected bioterror scenarios.

VSV-ΔG-Spike Candidate Vaccine Induces Protective Immunity and Protects K18-hACE2 Mice against SARS-CoV-2 Variants.

Since the emergence of the original SARS-CoV-2, several variants were described, raising questions as to the ability of recently developed vaccine platforms to induce immunity and provide protection against these variants. Here, we utilized the K18-hACE2 mouse model to show that VSV-ΔG-spike vaccination provides protection against several SARS-CoV-2 variants: alpha, beta, gamma, and delta. We show an overall robust immune response, regardless of variant identity, leading to reduction in viral load in target organs, prevention of morbidity and mortality, as well as prevention of severe brain immune response, which follows infection with various variants. Additionally, we provide a comprehensive comparison of the brain transcriptomic profile in response to infection with different variants of SARS-CoV-2 and show how vaccination prevents these disease manifestations. Taken together, these results highlight the robust VSV-ΔG-spike protective response against diverse SARS-CoV-2 variants, as well as its promising potential against newly arising variants.

Antiviral activity of glucosylceramide synthase inhibitors in alphavirus infection of the central nervous system.

Virus-induced CNS diseases impose a considerable human health burden worldwide. For many viral CNS infections, neither antiviral drugs nor vaccines are available. In this study, we examined whether the synthesis of glycosphingolipids, major membrane lipid constituents, could be used to establish an antiviral therapeutic target. We found that neuroinvasive Sindbis virus altered the sphingolipid levels early after infection in vitro and increased the levels of gangliosides GA1 and GM1 in the sera of infected mice. The alteration in the sphingolipid levels appears to play a role in neuroinvasive Sindbis virus replication, as treating infected cells with UDP-glucose ceramide glucosyltransferase (UGCG) inhibitors reduced the replication rate. Moreover, the UGCG inhibitor GZ-161 increased the survival rates of Sindbis-infected mice, most likely by reducing the detrimental immune response activated by sphingolipids in the brains of Sindbis virus-infected mice. These findings suggest a role for glycosphingolipids in the host immune response against neuroinvasive Sindbis virus and suggest that UGCG inhibitors should be further examined as antiviral therapeutics for viral infections of the CNS.

Anti-SARS-CoV-2 immunoadhesin remains effective against Omicron and other emerging variants of concern.

Blocking the interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with its angiotensin-converting enzyme 2 (ACE2) receptor was proved to be an effective therapeutic option. Various protein binders as well as monoclonal antibodies that effectively target the receptor-binding domain (RBD) of SARS-CoV-2 to prevent interaction with ACE2 were developed. The emergence of SARS-CoV-2 variants that accumulate alterations in the RBD can severely affect the efficacy of such immunotherapeutic agents, as is indeed the case with Omicron that resists many of the previously isolated monoclonal antibodies. Here, we evaluate an ACE2-based immunoadhesin that we have developed early in the pandemic against some of the recent variants of concern (VoCs), including the Delta and the Omicron variants. We show that our ACE2-immunoadhesin remains effective in neutralizing these variants, suggesting that immunoadhesin-based immunotherapy is less prone to escape by the virus and has a potential to remain effective against future VoCs.

Monkeypox DNA levels correlate with virus infectivity in clinical samples, Israel, 2022.

The current monkeypox virus global spread and lack of data regarding clinical specimens' infectivity call for examining virus infectivity, and whether this correlates with results from PCR, the available diagnostic tool. We show strong correlation between viral DNA amount in clinical specimens and virus infectivity toward BSC-1 cell line. Moreover, we define a PCR threshold value (Cq ≥ 35, ≤ 4,300 DNA copies/mL), corresponding to negative viral cultures, which may assist risk-assessment and decision-making regarding protective-measures and guidelines for patients with monkeypox.

Matrix Metalloproteinases Expression Is Associated with SARS-CoV-2-Induced Lung Pathology and Extracellular-Matrix Remodeling in K18-hACE2 Mice.

The COVID-19 pandemic caused by the SARS-CoV-2 infection induced lung inflammation characterized by cytokine storm and fulminant immune response of both resident and migrated immune cells, accelerating alveolar damage. In this work we identified members of the matrix metalloprotease (MMPs) family associated with lung extra-cellular matrix (ECM) destruction using K18-hACE2-transgenic mice (K18-hACE2) infected intranasally with SARS-CoV-2. Five days post infection, the lungs exhibited overall alveolar damage of epithelial cells and massive leukocytes infiltration. A substantial pulmonary increase in MMP8, MMP9, and MMP14 in the lungs post SARS-CoV-2 infection was associated with degradation of ECM components including collagen, laminin, and proteoglycans. The process of tissue damage and ECM degradation during SARS-CoV-2 lung infection is suggested to be associated with activity of members of the MMPs family, which in turn may be used as a therapeutic intervention.

rVSV-ΔG-SARS-CoV-2-S vaccine: repeated intramuscular (IM) toxicity, local tolerance, immunogenicity and biodistribution study in NZW rabbits.

BriLife®, a vector-based vaccine that utilizes the recombinant vesicular stomatitis virus (VSV) platform to express and present the spike antigen of SARS-CoV-2, is undergoing testing in a phase 2 clinical trial in Israel. A nonclinical repeated-dose (GLP) toxicity study in New Zealand white rabbits was performed to evaluate the potential toxicity, local tolerance, immunogenicity and biodistribution of the vaccine. rVSV-ΔG-SARS-CoV-2-S (or vehicle) was administered intramuscularly to two groups of animals (106, 107 PFU/animal, n = 10/sex/group) on three occasions, at 2-week intervals, followed by a 3-week recovery period. Systemic clinical signs, local reactions, body weight, body temperature, food consumption, ophthalmology, urinalysis, clinical pathology, C-reactive protein, viremia and antibody levels were monitored. Gross pathology was performed, followed by organs/tissues collection for biodistribution and histopathological evaluation. Treatment-related changes were restricted to multifocal minimal myofiber necrosis at the injection sites, and increased lymphocytic cellularity in the iliac and mesenteric lymph nodes and in the spleen. These changes were considered related to the inflammatory reaction elicited, and correlated with a trend for recovery. Detection of rVSV-ΔG-SARS-CoV-2-S vaccine RNA was noted in the regional iliac lymph node in animals assigned to the high-dose group, at both termination time points. A significant increase in binding and neutralizing antibody titers was observed following vaccination at both vaccine doses. In view of the findings, it was concluded that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe. These results supported the initiation of clinical trials.

Prolonged Protective Immunity Induced by Mild SARS-CoV-2 Infection of K18-hACE2 Mice.

Longevity of the immune response following viral exposure is an essential aspect of SARS-CoV-2 infection. Mild SARS-CoV-2 infection of K18-hACE2 mice was implemented for evaluating the mounting and longevity of a specific memory immune response. We show that the infection of K18-hACE2 mice induced robust humoral and cellular immunity (systemic and local), which persisted for at least six months. Virus-specific T cells and neutralizing antibody titers decreased over time, yet their levels were sufficient to provide sterile immunity against lethal rechallenge six months post-primary infection. The study substantiates the role of naturally induced immunity against SARS-CoV-2 infection for preventing recurring morbidity.

Neutralization of SARS-CoV-2 Variants by rVSV-ΔG-Spike-Elicited Human Sera.

The emergence of rapidly spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a major challenge to the ability of vaccines and therapeutic antibodies to provide immunity. These variants contain mutations of specific amino acids that might impede vaccine efficacy. BriLife® (rVSV-ΔG-spike) is a newly developed SARS-CoV-2 vaccine candidate currently in phase II clinical trials. It is based on a replication-competent vesicular stomatitis virus (VSV) platform. The rVSV-ΔG-spike contains several spontaneously acquired spike mutations that correspond to SARS-CoV-2 variants' mutations. We show that human sera from BriLife® vaccinees preserve comparable neutralization titers towards alpha, gamma, and delta variants and show less than a three-fold reduction in the neutralization capacity of beta and omicron compared to the original virus. Taken together, we show that human sera from BriLife® vaccinees overall maintain a neutralizing antibody response against all tested variants. We suggest that BriLife®-acquired mutations may prove advantageous against future SARS-CoV-2 VOCs.

Induction of Innate Immune Response by TLR3 Agonist Protects Mice against SARS-CoV-2 Infection.

SARS-CoV-2, a member of the coronavirus family, is the causative agent of the COVID-19 pandemic. Currently, there is still an urgent need in developing an efficient therapeutic intervention. In this study, we aimed at evaluating the therapeutic effect of a single intranasal treatment of the TLR3/MDA5 synthetic agonist Poly(I:C) against a lethal dose of SARS-CoV-2 in K18-hACE2 transgenic mice. We demonstrate here that early Poly(I:C) treatment acts synergistically with SARS-CoV-2 to induce an intense, immediate and transient upregulation of innate immunity-related genes in lungs. This effect is accompanied by viral load reduction, lung and brain cytokine storms prevention and increased levels of macrophages and NK cells, resulting in 83% mice survival, concomitantly with long-term immunization. Thus, priming the lung innate immunity by Poly(I:C) or alike may provide an immediate, efficient and safe protective measure against SARS-CoV-2 infection.

Preliminary nonclinical safety and immunogenicity of an rVSV-ΔG-SARS-CoV-2-S vaccine in mice, hamsters, rabbits and pigs.

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 108 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2.

ICAMs are dispensable for influenza clearance and anti-viral humoral and cellular immunity.

αLß2 (LFA-1) mediated interactions with ICAM-1 and ICAM-2 predominate leukocyte-vascular interactions, but their functions in extravascular cell-cell communications is still debated. The roles of these two ligands in leukocyte trafficking, lymphocyte differentiation, and immunity to influenza infections were dissected in the present study. Surprisingly, double ICAM-1 and ICAM-2 knock out mice (herein ICAM-1/2-/- mice) infected with a lab adapted H1N1 influenza A virus fully recovered from infection, elicited potent humoral immunity, and generated normal long lasting anti-viral CD8+ T cell memory. Furthermore, lung capillary ICAMs were dispensable for both NK and neutrophil entry to virus infected lungs. Mediastinal lymph nodes (MedLNs) of ICAM-1/2-/- mice poorly recruited naïve T cells and B lymphocytes but elicited normal humoral immunity critical for viral clearance and effective CD8+ differentiation into IFN-γ producing T cells. Furthermore, whereas reduced numbers of virus specific effector CD8+ T cells accumulated inside infected ICAM-1/2-/- lungs, normal virus-specific TRM CD8+ cells were generated inside these lungs and fully protected ICAM-1/2-/- mice from secondary heterosubtypic infections. B lymphocyte entry to the MedLNs and differentiation into extrafollicular plasmablasts, producing high affinity anti-influenza IgG2a antibodies, were also ICAM-1 and ICAM-2 independent. A potent antiviral humoral response was associated with accumulation of hyper-stimulated cDC2s in ICAM null MedLNs and higher numbers of virus-specific T follicular helper (Tfh) cells generated following lung infection. Mice selectively depleted of cDC ICAM-1 expression supported, however, normal CTL and Tfh differentiation following influenza infection, ruling out essential co-stimulatory functions of DC ICAM-1 in CD8+ and CD4+ T cell differentiation. Collectively our findings suggest that lung ICAMs are dispensable for innate leukocyte trafficking to influenza infected lungs, for the generation of peri-epithelial TRM CD8+ cells, and long term anti-viral cellular immunity. In lung draining LNs, although ICAMs promote lymphocyte homing, these key integrin ligands are not required for influenza-specific humoral immunity or generation of IFN-γ effector CD8+ T cells. In conclusion, our findings suggest unexpected compensatory mechanisms that orchestrate protective anti-influenza immunity in the absence of vascular and extravascular ICAMs.

SARS-CoV-2 receptor binding domain fusion protein efficiently neutralizes virus infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the COVID-19 pandemic. Currently, as dangerous mutations emerge, there is an increased demand for specific treatments for SARS-CoV-2 infected patients. The spike glycoprotein on the virus envelope binds to the angiotensin converting enzyme 2 (ACE2) on host cells through its receptor binding domain (RBD) to mediate virus entry. Thus, blocking this interaction may inhibit viral entry and consequently stop infection. Here, we generated fusion proteins composed of the extracellular portions of ACE2 and RBD fused to the Fc portion of human IgG1 (ACE2-Ig and RBD-Ig, respectively). We demonstrate that ACE2-Ig is enzymatically active and that it can be recognized by the SARS-CoV-2 RBD, independently of its enzymatic activity. We further show that RBD-Ig efficiently inhibits in-vivo SARS-CoV-2 infection better than ACE2-Ig. Mechanistically, we show that anti-spike antibody generation, ACE2 enzymatic activity, and ACE2 surface expression were not affected by RBD-Ig. Finally, we show that RBD-Ig is more efficient than ACE2-Ig at neutralizing high virus titers. We thus propose that RBD-Ig physically blocks virus infection by binding to ACE2 and that RBD-Ig should be used for the treatment of SARS-CoV-2-infected patients.

Monitoring Group Activity of Hamsters and Mice as a Novel Tool to Evaluate COVID-19 Progression, Convalescence, and rVSV-ΔG-Spike Vaccination Efficacy.

The COVID-19 pandemic initiated a worldwide race toward the development of treatments and vaccines. Small animal models included the Syrian golden hamster and the K18-hACE2 mice infected with SARS-CoV-2 to display a disease state with some aspects of human COVID-19. A group activity of animals in their home cage continuously monitored by the HCMS100 (Home cage Monitoring System 100) was used as a sensitive marker of disease, successfully detecting morbidity symptoms of SARS-CoV-2 infection in hamsters and in K18-hACE2 mice. COVID-19 convalescent hamsters rechallenged with SARS-CoV-2 exhibited minor reduction in group activity compared to naive hamsters. To evaluate the rVSV-ΔG-spike vaccination efficacy against SARS-CoV-2, we used the HCMS100 to monitor the group activity of hamsters in their home cage. A single-dose rVSV-ΔG-spike vaccination of the immunized group showed a faster recovery than the nonimmunized infected hamsters, substantiating the efficacy of rVSV-ΔG-spike vaccine. HCMS100 offers nonintrusive, hands-free monitoring of a number of home cages of hamsters or mice modeling COVID-19.

Increased lethality in influenza and SARS-CoV-2 coinfection is prevented by influenza immunity but not SARS-CoV-2 immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The continued spread of SARS-CoV-2 increases the probability of influenza/SARS-CoV-2 coinfection, which may result in severe disease. In this study, we examine the disease outcome of influenza A virus (IAV) and SARS-CoV-2 coinfection in K18-hACE2 mice. Our data indicate enhance susceptibility of IAV-infected mice to developing severe disease upon coinfection with SARS-CoV-2 two days later. In contrast to nonfatal influenza and lower mortality rates due to SARS-CoV-2 alone, this coinfection results in severe morbidity and nearly complete mortality. Coinfection is associated with elevated influenza viral loads in respiratory organs. Remarkably, prior immunity to influenza, but not to SARS-CoV-2, prevents severe disease and mortality. This protection is antibody-dependent. These data experimentally support the necessity of seasonal influenza vaccination for reducing the risk of severe influenza/COVID-19 comorbidity during the COVID-19 pandemic.

Implementation of Adenovirus-Mediated Pulmonary Expression of Human ACE2 in HLA Transgenic Mice Enables Establishment of a COVID-19 Murine Model for Assessment of Immune Responses to SARS-CoV-2 Infection.

HLA transgenic mice are instrumental for evaluation of human-specific immune responses to viral infection. Mice do not develop COVID-19 upon infection with SARS-CoV-2 due to the strict tropism of the virus to the human ACE2 receptor. The aim of the current study was the implementation of an adenovirus-mediated infection protocol for human ACE2 expression in HLA transgenic mice. Transient pulmonary expression of the human ACE2 receptor in these mice results in their sensitisation to SARS-CoV-2 infection, consequently providing a valuable animal model for COVID-19. Infection results in a transient loss in body weight starting 3 days post-infection, reaching 20-30% loss of weight at day 7 and full recovery at days 11-13 post-infection. The evolution of the disease revealed high reproducibility and very low variability among individual mice. The method was implemented in two different strains of HLA immunized mice. Infected animals developed strong protective humoral and cellular immune responses specific to the viral spike-protein, strictly depending on the adenovirus-mediated human ACE2 expression. Convalescent animals were protected against a subsequent re-infection with SARS-CoV-2, demonstrating that the model may be applied for assessment of efficacy of anti-viral immune responses.

Human Kidney Spheroids and Monolayers Provide Insights into SARS-CoV-2 Renal Interactions.

BACKGROUND: Although coronavirus disease 2019 (COVID-19) causes significan t morbidity, mainly from pulmonary involvement, extrapulmonary symptoms are also major componen ts of the disease. Kidney disease, usually presenting as AKI, is particularly severe among patients with COVID-19. It is unknown, however, whether such injury results from direct kidney infection with COVID-19's causative virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), or from indirect mechanisms. METHODS: Using ex vivo cell models, we sought to analyze SARS-CoV-2 interactions with kidney tubular cells and assess direct tubular injury. These models comprised primary human kidney epithelial cells (derived from nephrectomies) and grown as either proliferating monolayers or quiescent three-dimensional kidney spheroids. RESULTS: We demonstrated that viral entry molecules and high baseline levels of type 1 IFN-related molecules were present in monolayers and kidney spheroids. Although both models support viral infection and replication, they did not exhibit a cytopathic effect and cell death, outcomes that were strongly present in SARS-CoV-2-infected controls (African green monkey kidney clone E6 [Vero E6] cultures). A comparison of monolayer and spheroid cultures demonstrated higher infectivity and replication of SARS-CoV-2 in actively proliferating monolayers, although the spheroid cultures exhibited high er levels of ACE2. Monolayers exhibited elevation of some tubular injury molecules-including molecules related to fibrosis (COL1A1 and STAT6) and dedifferentiation (SNAI2)-and a loss of cell identity, evident by reduction in megalin (LRP2). The three-dimensional spheroids were less prone to such injury. CONCLUSIONS: SARS-CoV-2 can infect kidney cells without a cytopathic effect. AKI-induced cellular proliferation may potentially intensify infectivity and tubular damage by SARS-CoV-2, suggesting that early intervention in AKI is warranted to help minimize kidney infection.