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Current COVID-19 vaccines face certain limitations, which include waning immunity, immune escape by SARS-CoV-2 variants, limited CD8+cellular response, and poor induction of mucosal immunity. Here, we engineered a Clec9A-RBD antibody construct that delivers the Receptor Binding Domain (RBD) from SARS-CoV-2 spike protein to conventional type 1 dendritic cells (cDC1). We showed that single dose immunization with Clec9A-RBD induced high RBD-specific antibody titers with a strong T-helper 1 (TH1) isotype profile and exceptional durability, whereby antibody titers were sustained for at least 21 months post-vaccination. Uniquely, affinity maturation of the antibody response was observed over time, as evidenced by enhanced neutralization potency and breadth across the sarbecovirus family. Consistently and remarkably, RBD-specific T-follicular helper cells and germinal center B cells were still detected at 12 months post-immunization. Increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the immune sera was also measured over time with comparable efficacy against ancestral SARS-CoV-2 and variants, including Omicron. Furthermore, Clec9A-RBD immunization induced a durable poly-functional TH1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants, including Omicron, and with robust CD8+T cell signature. Lastly, Clec9A-RBD single dose systemic immunization primed effectively RBD-specific cellular and humoral mucosal immunity in lung. Taken together, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal immune responses against rapidly evolving SARS-CoV2 variants.

Bats are reservoir hosts for various zoonotic viruses with pandemdic potential in humans and livestock. In vitro systems for studying bat host-pathogen interactions are of significant interest. Here, we establish protocols to generate bat airway organoids (AOs) and airway epithelial cells differentiated at the air-liquid interface (ALI-AECs) from tracheal tissues of the cave-nectar bat Eonycteris spelaea. In particular, we describe steps which enable laboratories that do not have access to live bats to perform extended experimental work upon procuring an initial batch of bat primary airway tissue. Complete mucociliary differentiation required treatment with IL-13. E. spelaea ALI-AECs supported productive infection with PRV3M, an orthoreovirus for which Pteropodid bats are considered the reservoir species. However, these ALI-AECs did not support SARS-CoV-2 infection, despite E. spelaea ACE2 receptor being capable of mediating SARS-CoV-2 spike pseudovirus entry. This work provides critical model systems for assessing bat species-specific virus susceptibility and the reservoir likelihood for emerging infectious agents. Ministry of Education (MOE) Nanyang Technological University National Medical Research Council (NMRC) National Research Foundation (NRF) Published version This research was funded in part by Singapore National Research Foundation (NRF2012NRF-CRP001-056, NRF2016NRF-NSFC002-013), the Singapore National Medical Research Council of Singapore (MOH-COV-ID19RF2-0001, MOH-COVID19RF-003, MOH-OFIRG19-MAY-0011, MOH-OFIRG10NOV-0050) and the Ministry of Education Singapore (MOE2019-T2-2-130); Singapore National Medical Research Council Centre Grant Program – Diabetes, Tuberculosis and Neuroscience No.CGAug16M009; the NUS Reimagine Research Grant, Singapore; and the Nanyang Technological University Lee Kong Chian School of Medicine LEARN grant (021914-00001).LLYC is supported by the Lee Kong Chian School of Medi-cine, Nanyang Technological University under the Dean’s Postdoctoral Fellowship and the Wong Peng Onn Fellow-ship (002823-00001).

Background Multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) superspreading events suggest that aerosols play an important role in driving the coronavirus disease 2019 (COVID-19) pandemic. To better understand how airborne SARS-CoV-2 transmission occurs, we sought to determine viral loads within coarse (>5 μm) and fine (≤5 μm) respiratory aerosols produced when breathing, talking, and singing. Methods Using a G-II exhaled breath collector, we measured viral RNA in coarse and fine respiratory aerosols emitted by COVID-19 patients during 30 minutes of breathing, 15 minutes of talking, and 15 minutes of singing. Results Thirteen participants (59%) emitted detectable levels of SARS-CoV-2 RNA in respiratory aerosols, including 3 asymptomatic and 1 presymptomatic patient. Viral loads ranged from 63–5821 N gene copies per expiratory activity per participant, with high person-to-person variation. Patients earlier in illness were more likely to emit detectable RNA. Two participants, sampled on day 3 of illness, accounted for 52% of total viral load. Overall, 94% of SARS-CoV-2 RNA copies were emitted by talking and singing. Interestingly, 7 participants emitted more virus from talking than singing. Overall, fine aerosols constituted 85% of the viral load detected in our study. Virus cultures were negative. Conclusions Fine aerosols produced by talking and singing contain more SARS-CoV-2 copies than coarse aerosols and may play a significant role in SARS-CoV-2 transmission. Exposure to fine aerosols, especially indoors, should be mitigated. Isolating viable SARS-CoV-2 from respiratory aerosol samples remains challenging; whether this can be more easily accomplished for emerging SARS-CoV-2 variants is an urgent enquiry necessitating larger-scale studies.

Data on the viral loads in respiratory aerosols from patients infected with Delta and Omicron variants are limited. In this study, we used an exhaled breath bioaerosol collector to collect aerosol samples in coarse (> 5µm) and fine (≤ 5µm) fractions from COVID-19 patients infected with these VOCs while doing various respiratory activities. Samples were tested via SARS-CoV-2 RT-qPCR and virus culture. Nine patients (4 Delta and 5 Omicron) were included. Viral RNA was detectable in seven participants, with greater viral loads in fine aerosols. Notably SARS-CoV-2 RNA was consistently detectable in respiratory samples of all Omicron patients despite them being fully vaccinated and mostly asymptomatic in contrast with Delta patients. Singing and talking without mask generated the greatest viral loads underscoring the transmission potential of SARS-CoV-2 and its variants via respiratory aerosols. The more consistent detection of viral RNA in Omicron-infected patients may account for its greater transmissibility.

BackgroundOn 26 November 2021, the World Health Organization designated the B.1.1.529 lineage of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) as the fifth variant of concern, Omicron. Infections have quickly spread worldwide, but understanding of the viral dynamics and the cytokine and cellular immunological response during infection remain limited.MethodsDetailed patient-level data from 174 age-matched patients with sequence confirmed Omicron or Delta infection admitted to the National Centre for Infectious Diseases, Singapore were analyzed in an observational cohort study. Peripheral blood samples for measurement of SARS-CoV-2 immunological parameters were obtained from a subset. Respiratory samples were collected for viral cultures and correlated to corresponding PCR cycle threshold (Ct) values. ResultsOmicron and Delta variant infections in this hospitalized cohort were mild with only 3 (3%) and 14 (16%) developing pneumonia respectively. Omicron infections were more likely to present with sore throat (46.0 vs x23.0%, p=0.005). Neutrophil counts and C-reactive protein (CRP) were significantly lower among the Omicron cohort (Median neutrophil 2.95 [IQR 2.16 – 3.96] vs 4.60 [IQR 3.76 – 6.10] x 109/L , pConclusionsOmicron infections resulted in mild vaccine breakthrough illness in the majority of patients. Compared with Delta, Omicron infections were more frequently associated with upper respiratory tract infections, had lower viral loads, lower levels of pro-inflammatory cytokines and less dysregulated immune cell profiles.

The ongoing SARS-CoV-2 pandemic has tested the capabilities of public health and scientific community. Since the dawn of the twenty-first century, viruses have caused several outbreaks, with coronaviruses being responsible for 2: SARS-CoV in 2007 and MERS-CoV in 2013. As the border between wildlife and the urban population continue to shrink, it is highly likely that zoonotic viruses may emerge more frequently. Furthermore, it has been shown repeatedly that these viruses are able to efficiently evade the innate immune system through various strategies. The strong and abundant antiviral innate immunity evasion strategies shown by SARS-CoV-2 has laid out shortcomings in our approach to quickly identify and modulate these mechanisms. It is thus imperative that there be a systematic framework for the study of the immune evasion strategies of these viruses, to guide development of therapeutics and curtail transmission. In this review, we first provide a brief overview of general viral evasion strategies against the innate immune system. Then, we utilize SARS-CoV-2 as a case study to highlight the methods used to identify the mechanisms of innate immune evasion, and pinpoint the shortcomings in the current paradigm with its focus on overexpression and protein-protein interactions. Finally, we provide a recommendation for future work to unravel viral innate immune evasion strategies and suitable methods to aid in the study of virus-host interactions. The insights provided from this review may then be applied to other viruses with outbreak potential to remain ahead in the arms race against viral diseases.

BackgroundMultiple SARS-CoV-2 superspreading events suggest that aerosols play an important role in driving the COVID-19 pandemic. However, the detailed roles of coarse (>5μm) and fine (≤5μm) respiratory aerosols produced when breathing, talking, and singing are not well-understood.MethodsUsing a G-II exhaled breath collector, we measured viral RNA in coarse and fine respiratory aerosols emitted by COVID-19 patients during 30 minutes of breathing, 15 minutes of talking, and 15 minutes of singing.ResultsAmong the 22 study participants, 13 (59%) emitted detectable levels of SARS-CoV-2 RNA in respiratory aerosols, including 3 asymptomatic patients and 1 presymptomatic patient. Viral loads ranged from 63–5,821 N gene copies per expiratory activity per patient. Patients earlier in illness were more likely to emit detectable RNA, and loads differed significantly between breathing, talking, and singing. The largest proportion of SARS-CoV-2 RNA copies was emitted by singing (53%), followed by talking (41%) and breathing (6%). Overall, fine aerosols constituted 85% of the viral load detected in our study. Virus cultures were negative.ConclusionsFine aerosols produced by talking and singing contain more SARS-CoV-2 copies than coarse aerosols and may play a significant role in the transmission of SARS-CoV-2. Exposure to fine aerosols should be mitigated, especially in indoor environments where airborne transmission of SARS-CoV-2 is likely to occur. Isolating viable SARS-CoV-2 from respiratory aerosol samples remains challenging, and whether this can be more easily accomplished for emerging SARS-CoV-2 variants is an important enquiry for future studies.Key PointsWe sampled respiratory aerosols emitted by COVID-19 patients and discovered that fine aerosols (≤5μm) generated during talking and singing contain more SARS-CoV-2 copies than coarse aerosols (>5μm) and may play a significant role in the transmission of SARS-CoV-2.