BROAD ANTIBODIES TO FUNCTIONALLY CONSTRAINED REGIONS OF SARS-CoV-2 SPIKE

Background: While remarkable and rapid progress was made in fighting the SARS-CoV-2 pandemic with vaccines and therapeutic antibodies, these approaches were quickly compromised by viral evolution. Therapeutic monoclonal antibodies (mAbs) that were once authorized for clinical use, which all target the receptor binding domain (RBD), are no longer effective against current variants of concern (VOCs) due to mutations in this region of Spike. Thus, to achieve durable protection against SARS-CoV-2, novel mAbs need to show breadth and potency across VOCs and target epitopes that are more constrained. Method(s): mAbs from an individual who had a breakthrough Delta VOC infection after vaccination were isolated from Spike-specific memory B cells. mAbs were assessed for binding affinity and neutralization potency using Spike-pseudotyped lentivirus (PSV) and live SARS-CoV-2 virus neutralization assays. Epitopes were mapped using deep mutational sequencing (DMS) and structural-based methods. Result(s): Three novel mAbs (C68.3, C68.13, C68.59) demonstrated binding breadth to Spikes from various VOCs including Omicron VOCs despite that C68 had not yet been exposed to Omicron. These mAbs potently neutralized the Wuhan-Hu-1 vaccine and Delta strains (IC50 = 9-61ng/mL), and early Omicron strains BA.1, BA.2, BA.5 (IC50 = 12-149 ng/mL). C68.3 and C68.59 retained potency against recent VOCs BQ.1.1 and XBB (IC50 = 121-122 ng/mL and 56-82 ng/mL, respectively) in the PSV assay. Similar neutralization activity was observed in the live virus assay. The potency of these mAbs was greater against Omicron VOCs than all but one of the mAbs previously authorized for treatment and they showed greater breadth. The mAbs target distinct epitopes on the Spike glycoprotein, two in the RBD (C68.3, C68.13) and one in an invariant region downstream of RBD in subdomain 1 (SD1) (C68.59). Structural analysis of C68.59 Fab binding to Spike trimer revealed significant allosteric changes to regions of Spike outside of the epitope in the S2 unit. Finally, DMS escape pathways showed these mAbs target regions highly conserved across VOCs that are also functionally constrained, suggesting escape could incur a fitness cost. Conclusion(s): Overall, these mAbs are novel in their breadth across VOCs and include a potent mAb targeting a rare epitope outside of the RBD in SD1. These mAbs focus on diverse, functionally constrained regions in Spike making them candidates for development as combination therapeutics with good durability against future VOCs.

[Analysis on the household secondary attack rates of the SARS-CoV-2 Delta variant and the associated factors].

Objective: To evaluate the household secondary attack rates of the SARS-CoV-2 Delta variant and the associated factors. Methods: A COVID-19 outbreak caused by the Delta variant occurred in Nanjing in July 2021. A total of 235 cases with current addresses in Nanjing were reported from 171 households. The subjects in this study were selected from household close contact(s) of infected cases. The information on household index cases and their contacts were collected, and the household secondary attack rate (HSAR) and the risk factors were analyzed by the multi-factor logistic regression model. Results: A total of 234 cases of household close contacts and 64 household secondary cases were reported from 103 households, and the HSAR was 27.4% (64/234, 95%CI:22.0% to 33.4%). The proportions of household size for 2 to 3, 4 to 5, and 6 to 9 were 64.1% (66), 26.2% (27) and 9.7% (10), respectively. A total of 35 cases of household cluster outbreaks were reported (35/103, 34.0%). The number of the first case in the household (FCH) was 103 and males accounted for 27.2% (28 cases), with the median age (Q1, Q3) of 49 (9, 56). The number of household close contacts was 234 and males accounted for 59.0% (138 cases), with the median age (Q1, Q3) of 42 (20, 55) and the median exposure period (Q1, Q3) of 3 (1, 3) days. The multi-factor logistic regression model showed that the higher HSAR was observed in the FCH with the features of airport staff (OR=2.913, 95%CI:1.469-5.774), detection from home quarantine screening (OR=6.795, 95%CI:1.761-26.219) and detection from mass screening (OR=4.239, 95%CI:1.098-16.368). Meanwhile, higher HSAR was observed in cases with longer household exposure (OR=1.221, 95%CI:1.040-1.432), non-vaccination (OR=2.963, 95%CI:1.288-6.813) and incomplete vaccinations (OR=2.842, 95%CI:0.925-8.731). Conclusion: The generation interval of the Delta variant is shortened, and the ability of transmission within the household is enhanced. In the outbreak in Nanjing, the associated factors of HSAR are occupation, detection route, vaccination and exposure period.

[Kinetics of SARS-CoV-2-specific antibodies among inactivated COVID-19 vaccine recipients, SARS-CoV-2 natural infection cases, and breakthrough cases].

Between August and September, 2021, this study included 605 SARS-CoV-2 natural infection cases and 589 SARS-CoV-2 breakthrough cases from Nanjing and Yangzhou, as well as 690 inactivated COVID-19 vaccine recipients from Changzhou, China. In SARS-CoV-2 natural infection cases, the age range was 19-91 years (median age: 66 year), and the medians(Q1,Q3) of IgG titers were 0.19 (0.06-1.31), 3.70 (0.76-69.48), 15.31 (2.59-82.16), 4.41 (0.99-31.74), 2.31 (0.75-13.83), 2.28 (0.68-9.94) and 2.80 (1.00-9.53) at one to seven weeks after SARS-CoV-2 infection, respectively. In SARS-CoV-2 breakthrough cases, the age range was 18-76 years (median age: 45 year), and the medians(Q1,Q3)of IgG titers were 1.93 (0.34-26.67), 38.87 (7.90-121.0), 75.09 (11.85-123.70), 21.97 (5.20-95.58), 13.97 (3.47-46.82), 9.56 (2.48-33.38) and 4.38 (1.87-11.00) at one to seven weeks after SARS-CoV-2 infection, respectively. In inactivated COVID-19 vaccine recipients, the age range was 18-87 years (median age: 47 years), and the medians(Q1,Q3)of IgG titers were 16.22 (15.84-33.42), 5.35 (2.96-13.23), 3.30 (2.18-6.18), 3.14 (1.16-5.70), 2.77 (1.50-4.52), 2.72 (1.76-4.36), 2.01 (1.27-3.51) and 1.94 (1.35-3.09) at one to eight months after SARS-CoV-2 infection, respectively. The results suggested that IgG antibodies increased gradually within two weeks after SARS-CoV-2 infection, then declined gradually at three to seven weeks in SARS-CoV-2 natural infection cases. In SARS-CoV-2 breakthrough cases, IgG antibodies increased rapidly within two weeks, then declined gradually at three to seven weeks after SARS-CoV-2 infection. Additionally, IgG antibodies decreased rapidly within three months, then decreased gradually and remained at a low level within three months after immunization.

Implementation and Experience of a Mixed Teaching Mode of Preventive Medicine Course Based on Network Teaching Platform under the Epidemic Situation of COVID-19 Pneumonia

With the rapid development of computer technology, the traditional teaching mode has been changed. Computer technology has played a decisive role in promoting teaching reform. This paper mainly discusses the implementation and experience of the mixed teaching mode of preventive medicine course based on the network teaching platform under the epidemic situation of novel coronavirus pneumonia. The mixed teaching mode of "online live teaching + MOOC + TBL"was used for the study on the online course of preventive medicine, and a mixed teaching mode of preventive medicine course based on the network teaching platform was constructed. In the teaching practice for clinical medical students, it was found that this teaching model could help to improve their teaching satisfaction, performance, autonomous learning ability, and ability to obtain information and evaluate information, through which a satisfactory teaching result has been achieved. The results indicate that this teaching mode may be an efficient and innovative teaching mode, and an effective way to improve the quality of education and teaching, worth popularizing. © 2022 Owner/Author.

EXPRESSION OF EPITHELIAL PROTEASES PREDICTS SARS-COV-2 ENTRY IN HUMAN ENTEROIDS

Introduction: Coronavirus Disease 2019 (COVID-19) is an ongoing public health crisis that has sickened or precipitated death in millions. The etiologic agent of COVID-19, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), infects the intestinal epithelium and can persist long after the respiratory infection has cleared. We previously observed that intestinal SARS-CoV-2 infection levels varied by individual donors and did not correlate positively with ACE2, the cognate SARS-CoV-2 receptor. Therefore we aimed to delineate host factors that influence viral infection in the intestine. Methods: Published dataset GSE75214 was downloaded and expression levels of select genes were querried. Primary human ileal spheroids (enteroids), derived from healthy donors and patients with Crohn's disease (CD), were grown on 2D transwells until confluent. Cells were differentiated for 3d before infection with a modified vesicular stomatitis virus expressing the SARS-CoV-2 spike protein (VSV-SARS-CoV-2) and GFP for 1h at a multiplicity of infection of ~0.5. Cells were harvested pre-infection and 24h after infection and expression of select genes was performed by qRT-PCR. Expression data were fit to a linear regression model to predict viral RNA levels. Results: Small intestine biopsy samples from CD patients demonstrated a reduction in ACE and an increase in CTSB and CTSL expression during active inflammation compared to healthy controls. Viral RNA expression did not correlate with ACE2 expression in CD enteroids. A subset of CD enteroids exhibited enhanced protease expression (TMPRSS2, TMPRSS4, CTSL), each of which correlated with higher viral RNA levels (P=0.04, P=0.002, P=0.006, respectively). Expression of these proteases was higher in the pre-infection for the sample subset. Principle component analysis of uninfected expression data demonstrated these samples clustered separately from the others, with the difference driven by TMPRSS2, TMPRSS4, and CTSL. Modeling viral RNA levels based on gene expression revealed expression levels of these proteases are a predictive expression signature. Conclusions: Host protease expression can predict SARS-CoV-2 infection and represent potential therapeutic targets for COVID-19. This is consistent with the recent report showing that cathepsin inhibition reduces SARS-CoV-2 spike-mediated syncytia formation. High expression of these proteases in the intestine may also be a novel biomarker for the risk of intestinal complications associated with COVID-19.(Figure Presented)RNA data from dataset GSE75214 demonstrating reduced ACE2 and increased CTSB and CTSL in patients with Crohn's disease during active inflammation compared to healthy controls. (Figure Presented) Enteroids from healthy control donors and patients with Crohn's disease were grown in 2D transwells and expression of indicated genes was assessed in pre-infection (A) and after infection with VSV-SARS-CoV-2 (B)

SARS-CoV-2 VARIANTS INCREASE KINETIC STABILITY of OPEN SPIKE CONFORMATIONS

Background: SARS-CoV-2 variants of concern harbor mutations in the Spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses, with structural studies of soluble S variants revealing an increased propensity towards conformations accessible to the human Angiotensin-Converting Enzyme 2 (hACE2) receptor. However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Methods: Here, we report single-molecule Förster Resonance Energy Transfer (smFRET) studies of S variants of concern containing critical mutations, including D614G and E484K, in the context of virus particles. Results: Investigated variants were shown by smFRET to predominantly occupy more open hACE2-accessible conformations, agreeing with predictions from structures of soluble trimers. Additionally, S variants exhibited decelerated transitions from hACE2-accessible/bound states. Conclusion: Here, we provide the real-time dimension to distinct structures of Spikes in the context of virus particles and present the first experimental evidence of increased stability of Spike variants. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.

ALTERED FEATURES of B and T CELL RESPONSES to BNT162 VACCINE in HEMODIALYSIS PATIENTS

Background: People receiving hemodialysis (HD) are highly vulnerable to SARS-CoV-2 infection and develop lower antibody responses to SARS-CoV-2 mRNA vaccines. However, the underlying immune defects are poorly understood. We compared immune responses of 27 HD patients with 22 health care workers (HCW) before and up to 4 months after 2 doses of BNT162 SARS-CoV-2 vaccination. All participants were confirmed to be SARS-CoV-2 naïve. Methods: We profiled B cells, CD4 T cells, CD8 T cells and humoral responses and examined associations between these arms of immunity. We used high-parameter flow cytometry to study: i) receptor binding domain (RBD)-specific B cells;ii) the phenotype of Spike (S)-specific CD4 and CD8 T cells identified by an activation-induced marker (AIM) assay;iii) effector functions of S-specific CD4 and CD8 T cells by intracellular staining (ICS). We measured humoral responses by ELISA RBD. Results: In each cohort, two vaccine doses enhanced RBD-specific B cell responses, with a significantly greater increase after the second dose (V2 than after the first dose (V1). Their magnitude was significantly lower in HD than in HCW at V1 (p=0.002) and V2 (p=0.002), which was consistent with the detection of lower anti-RBD IgG antibody levels at the same time points (V1: p<0.001;V2: p<0.001). The subsequent rates of B cell decline were similar in HD and HCW. As CD4 help is critical for B cell and CD8 T cell immunity, we compared Spike (S)-specific T cells responses between cohorts. While we observed no significant quantitative difference in the magnitude of vaccine-specific CD4 T cells between HD and HCW at V2, phenotypic and functional Thelper profiles differed significantly. The frequency of vaccine-specific CXCR3+ Th1 CD4 T cells was significantly increased in HD compared to HCW (p=0.008), and TNFα+ CD4 T cell responses were elevated in HD (p=0.01). In contrast to CD4 T cells, S-specific CD8 T cell responses were quantitatively reduced in HD compared to HCW after each dose (V1: p<0.001;V2: p<0.001). Conclusion: People on HD develop poor B cell and CD8 T cell responses after SARS-CoV-2 mRNA vaccination. These defects are associated with a skewed differentiation of vaccine-specific CD4 T cells toward CXCR3+ and TNFα+ Th1-like profiles, and probable altered crosstalk between Thelper and B cells. Further study is needed to determine if impaired B and T cell vaccine immunity in addition to defective antibody responses increases vulnerability of HD patients to breakthrough COVID-19 infection.

SARS-CoV-2 SPIKE EXPRESSION at the SURFACE of INFECTED HUMAN AIRWAY EPITHELIAL CELLS

Background: Emerging evidence points out to potential benefits from Fc-mediated effector functions in SARS-CoV-2 infection. Some Fc-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP) require recognition of the antigen at the surface of infected cells. Methods: To evaluate the expression levels of SARS-CoV-2 Spike at the surface of infected airway epithelial cells, we developed an intracellular staining against SARS-CoV-2 nucleocapsid (N). This assay allows the distinction between infected versus uninfected cells. Human primary airway epithelial cells (pAECs) were infected with authentic SARS-CoV-2 D614G or Alpha variants. Infected cells were identified with an anti-N antibody and cell surface expression of Spike measured with the conformational-independent anti-S2 CV3-25 antibody. Results: We found robust SARS-CoV-2 Spike expression at the cell surface of pAECs. Infected cells were readily recognized with plasma from convalescent and vaccinated individuals. Importantly, recognition of SARS-CoV-2 infected cells strongly correlated with Fc-mediated effector functions measured in a cohort of vaccinated naïve and previously-infected individuals. Conclusion: Altogether, our findings further support the importance of measuring Fc-mediated effector function in infection and vaccination settings for SARS-CoV-2.

A FC-ENHANCED NON-NEUTRALIZING ANTIBODY DELAYS SARS-CoV-2-INDUCED DEATH in MICE

Background: Both neutralizing activity and Fc-mediated effector functions of antibodies are believed to contribute to protection against SARS-CoV-2. However, it is unclear if antibody effector functions alone could protect against SARS-CoV-2 infection. Methods: We isolated CV3-13 from a convalescent individual with potent Fc-mediated effector functions. Neutralization capacity of this antibody was measured by both a pseudovirus neutralization assay and an authentic virus microneutralization assay. We mutated the Fc-portion of CV3-13 to enhance (GASDALIE) or reduce (LALA) its capacity to mediate antibody dependant cellular cytotoxicity (ADCC). Structural analysis of CV3-13 was done by cryo-EM to characterize its epitope and its angle of approach. Finally, CV3-13 and CV3-13 GASDALIE were used in vivo in a K18-hACE2 transgenic mouse model challenged with SARS-CoV-2-nLuc to see if they altered viral replication and/or contributed to protection against SARS-CoV-2. Results: While CV3-13 did not neutralize SARS-CoV-2, it demonstrated nanomolar affinity towards the SARS-CoV-2 Spike and mediated strong ADCC. The cryo-EM structure of CV3-13 in complex with the SARS-CoV-2 Spike revealed that the antibody bound to a novel NTD epitope that partially overlapped with a frequently mutated NTD supersite in SARS-CoV-2 variants. Interestingly, this angle of approach was not observed for previously described NTD-directed antibodies. While CV3-13 did not alter the replication dynamics of SARS-CoV-2 in a K18-hACE2 transgenic mouse model, a Fc-enhanced CV3-13 significantly delayed neuroinvasion and death in prophylactic settings. Conclusion: CV3-13 represents a new class of non-neutralizing NTD-directed mAbs that can mediate Fc-effector functions both in vitro and in vivo. While effector functions alone did not protect K18-hACE2 mice from SARS-CoV-2-nLuc challenge, our data indicate that along with neutralization, additional antibody properties including Fc-mediated effector functions contribute to limiting viral spread and aid in fighting SARS-CoV-2 infection.

The Effect of Ivermectin on Reducing Viral Symptoms in Patients with Mild COVID-19

Ivermectin is widely prescribed as a potential treatment for coronavirus disease 2019, despite uncertainty about its clinical benefit. To determine whether ivermectin is an efficacious treatment for mild coronavirus disease 2019 is the objective of the study. A total of 476 adult patients with mild symptoms for 7 d or fewer in Jinan, China, were enrolled and followed up. Patients were randomly selected to receive ivermectin, 300 μg/kg body weight per day for 5 d or placebo. The median time to resolution of symptoms was 10 d (interquartile range, 9-13) in the ivermectin group whereas it was 12 d (interquartile range, 9-13) in the placebo group (hazard ratio for resolution of symptoms, 1.07 [95 % confidence interval, 0.87 to 1.32];p=0.53 by log-rank test). By d 21, 82 % in the ivermectin group and 79 % in the placebo group had resolved symptoms. The most common solicited adverse event was headache, reported by 104 patients (52 %) who received ivermectin and 111 patients (56 %) who received placebo. The most serious adverse event was multiorgan failure. Among adults with mild coronavirus disease 2019, a 5 d course of ivermectin, compared with placebo, did not significantly improve the time to resolution of symptoms. The findings do not support the use of ivermectin for treatment of mild coronavirus disease 2019. © 2022 Indian Pharmaceutical Association. All rights reserved.

Evaluating the Anxiety and Adjustment of Physical Education Teachers Implementing Flipped Class Based on Data Visualization Technology

Data visualization technology uses graphics and images to intuitively express the influence of teachers' implementation of flipped physical education. The sample comes from a flipped physical education classroom conducted by 130 physical education teachers (N=130, age 28-55) from 6 university sports departments in China. The research results show that gender, teaching age, and education level have a greater impact on teachers' implementation of flipped physical education classes, and there are significant differences in anxiety levels. During the COVID-19 period, how to implement physical education has caused a lot of troubles and concerns. Teachers generally believed that they had lost their status as physical education teachers, and very worried about how toimplement physical education In quarantine state. However, after a period of trying to flip physical education, teachers' attitudes have changed significantly. Most physical education teachers hold a positive attitude. This is because their worries and confusion about flipping the physical education classroom transition to knowing the reason, and increasing the awareness and initiative of physical education teachers in teaching. © 2021 Polish Academy of Sciences. All rights reserved.

Regulatory considerations for using surrogate endpoints to accelerate the development of COVID-19 vaccines

In the research and development (R&D) of vaccines, surrogate endpoints have attracted more and more attention. Recently, using surrogate endpoints to accelerate vaccine R&D and marketing has become a potential direction in the future due to the intensification of the COVID-19 pandemic, the frequent occurrence of new coronavirus mutants, and the shortage of vaccine supply in the international market. This article summarized the clinical endpoints of the currently marketed new coronavirus (SARS-CoV-2) vaccines and explored the way to use surrogate endpoints to evaluate the efficacy of vaccines. In the context of the increasingly severe COVID-19 pandemic, this article provides practicable regulatory suggestions for future vaccine R&D and review to select surrogate endpoints to accelerate the approval of vaccines.

Developing Multifunctional/Smart Civil Engineering Materials to Fight Viruses

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed an extraordinary threat to global public health, wealth, and well-being. As the carriers of human life and production, infrastructures need to be upgraded to mitigate and prevent the spread of viral diseases. Developing multifunctional/smart civil engineering materials to fight viruses is a promising approach to achieving this goal. In this Perspective, a basic introduction on viruses and their structure is provided. Then, the current design principles of antiviral materials and structures are examined. Subsequently, the possibility of developing active/passive antiviral civil engineering materials (including cementitious composites, ceramics, polymers, and coatings) is proposed and envisioned. Finally, future research needs and potential challenges to develop antiviral civil engineering materials are put forward. The proposed strategies to develop multifunctional/smart antiviral civil engineering materials will aid in the construction of smart infrastructures to prevent the spread of viruses, thus improving human life and health as well as the sustainability of human society.

An Improved Agent-based Model Using Discrete Event Simulation for Nonpharmaceutical Interventions

The traditional agent-based model requires high computing power of the central processing unit. Thus, an improved agent-based model combined with the discrete event simulation method is proposed. The result of the equation-based Susceptible-Exposed-Infective-Asymptomatic-Recovered (SEIAR) model with the same parameter combination, which has been demonstrated to be effective, is used to verify the validity of this improved agent-based model. Additionally, an analysis based on simulation results of the Contact Tracing Measure (CTM), Location-Based Checking-Testing Measure (LCTM), Lockdown Measure (LM), Mobile Cabin Isolation and Hospital Measure (MCHM) is presented. The simulation results show that implementing long-term lockdown measures has the best effect on epidemic control. Moreover, according to the simulation results, we inferred that using only nonpharmaceutical epidemic prevention measures may result in a second outbreak of COVID-19 owing to the risk of asymptomatic transmission. Author

An ENV-GAG MRNA vaccine protects macaques from heterologous TIER-2 SHIV infection

Background: The development of a preventive vaccine remains a critical priority for ending the HIV/AIDS pandemic. Critical improvements in mRNA technology, as attested by recent successes in preventing COVID-19 disease, led us to develop an mRNA platform for HIV vaccines. Methods: In this regard, we designed an mRNA vaccine with different HIV-1 envelope mRNAs from 3 different clades co-formulated with SIV gag mRNA, which can assemble virus like particles (VLPs) in vivo. Rhesus macaques were primed with a transmitted-founder clade-B Env lacking the 276 N-glycan followed by multiple glycan-repaired autologous and bivalent heterologous (clades A and C) booster immunizations. Results: Immunized animals rapidly developed autologous neutralizing antibodies and eventually, after the second heterologous boost, cross-reactive tier-2 neutralizing antibodies, albeit at low titers. Vaccinated animals were protected from repeated low-dose rectal challenges with a heterologous tier-2 simian-human immunodeficiency virus (AD8). Protection was correlated with the presence of antibodies to the CD4-binding site. Conclusion: Thus, the Gag-Env VLP mRNA platform offers a promising strategy for the development of an HIV-1 vaccine.

Increased cathepsin expressioncorrelates with sars-cov-2infection in human ibd enteroids

Introduction Coronavirus Disease 2019 (COVID-19) is an ongoing publichealth crisis that has sickened or precipitated death in millions.The etiologic agent of COVID-19, Severe Acute RespiratorySyndrome Coronavirus 2 (SARS-CoV-2), infects the intestinalepithelium, with viral RNA shed in the stool, and can induce GIsymptoms similar to the human inflammatory bowel diseases(IBD). An international surveillance epidemiology study,SECURE-IBD, reported that the standardized mortality ratiotrends higher in IBD patients (1.5-1.8) and that 5-aminosalicylicacid (5-ASA) therapy correlates with poor outcome. Togetherthese data indicate patients with IBD may represent aparticularly vulnerable population during this COVID-19pandemic. Methods Published datasets GSE75214 and GSE16879 were downloadedand expression levels of select genes were querried usingRStudio. Primary human ileal spheroids (enteroids), derivedfrom healthy donors and patients with Crohn's disease (CD),were grown on 2D transwells until confluent. Cells were thendifferentiated for 3d before infection with a modified vesicularstomatitis virus expressing the SARS-CoV-2 spike protein (VSV-SARS-CoV-2) and green fluorescent protein (GFP) for 1 h at amultiplicity of infection (MOI) of ∼0.5. Healthy enteroids weretreated with 10 ng/ml of human Tumor Necrosis Factor alpha (TNF-α) for 24h before infection via the basolateral reservoir or5-ASA 5h before infection via the apical reservoir. 24h afterinfection, cells were processed for immunofluorescence or RNAexpression of select genes by qRT-PCR. Results VSV-SARS-CoV-2 was able to infect both healthy and CDenteroids as determined by co-staining of GFP, indicative ofvirus infection, and the viral receptor ACE2. However, levels ofGFP fluorescence did not correlate with ACE2 expression in CDenteroids. A subset of CD enteroids exhibited enhanced proteaseexpression (TMPRSS2, TMPRSS4, CTSL), each of whichcorrelated with higher viral RNA levels (P=0.04, P=0.002,P=0.006, respectively). In Vero E6 cells, 5-ASA inhibited thereplication of a clinical isolate of SARS-CoV-2 in aconcentration-dependent manner. Treating healthy enteroidswith 5-ASA did not have any effect on viral proliferation, whileTNF-α pretreatment reduced viral RNA. 5-ASA treatment causeda reduction of ACE2 and an increase in CTSL expression. Conclusions Host proteases, particularly the lysosomal protease CTSL,contribute to the infection of CD enteroids and may representnovel therapeutic targets in patients with IBD and COVID-19. 5-ASA modulates the expression of several epithelial genesrelevant to SARS-CoV-2 infection, yet does not alter viralreplication in healthy enteroids.