SARS-CoV-2-specific T cell immunity in cases of COVID-19 and SARS, and uninfected controls.

Memory T cells induced by previous pathogens can shape susceptibility to, and the clinical severity of, subsequent infections1. Little is known about the presence in humans of pre-existing memory T cells that have the potential to recognize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here we studied T cell responses against the structural (nucleocapsid (N) protein) and non-structural (NSP7 and NSP13 of ORF1) regions of SARS-CoV-2 in individuals convalescing from coronavirus disease 2019 (COVID-19) (n = 36). In all of these individuals, we found CD4 and CD8 T cells that recognized multiple regions of the N protein. Next, we showed that patients (n = 23) who recovered from SARS (the disease associated with SARS-CoV infection) possess long-lasting memory T cells that are reactive to the N protein of SARS-CoV 17 years after the outbreak of SARS in 2003; these T cells displayed robust cross-reactivity to the N protein of SARS-CoV-2. We also detected SARS-CoV-2-specific T cells in individuals with no history of SARS, COVID-19 or contact with individuals who had SARS and/or COVID-19 (n = 37). SARS-CoV-2-specific T cells in uninfected donors exhibited a different pattern of immunodominance, and frequently targeted NSP7 and NSP13 as well as the N protein. Epitope characterization of NSP7-specific T cells showed the recognition of protein fragments that are conserved among animal betacoronaviruses but have low homology to 'common cold' human-associated coronaviruses. Thus, infection with betacoronaviruses induces multi-specific and long-lasting T cell immunity against the structural N protein. Understanding how pre-existing N- and ORF1-specific T cells that are present in the general population affect the susceptibility to and pathogenesis of SARS-CoV-2 infection is important for the management of the current COVID-19 pandemic.

Study protocol: infectious diseases consortium (I3D) for study on integrated and innovative approaches for management of respiratory infections: respiratory infections research and outcome study (RESPIRO).

BACKGROUND: Community-acquired respiratory infections are a leading cause of illness and death globally. The aetiologies of community-acquired pneumonia remain poorly defined. The RESPIRO study is an ongoing prospective observational cohort study aimed at developing pragmatic logistical and analytic platforms to accurately identify the causes of moderate-to-severe community-acquired pneumonia in adults and understand the factors influencing disease caused by individual pathogens. The study is currently underway in Singapore and has plans for expansion into the broader region. METHODS: RESPIRO is being conducted at three major tertiary hospitals in Singapore. Adults hospitalised with acute community-acquired pneumonia or lower respiratory tract infections, based on established clinical, laboratory and radiological criteria, will be recruited. Over the course of the illness, clinical data and biological samples will be collected longitudinally and stored in a biorepository for future analysis. DISCUSSION: The RESPIRO study is designed to be hypothesis generating, complementary to and easily integrated with other research projects and clinical trials. The detailed clinical database and biorepository will yield insights into the epidemiology and outcomes of community-acquired lower respiratory tract infections in Singapore and the surrounding region and offers the opportunity to deeply characterise the microbiology and immunopathology of community-acquired pneumonia.

Feasibility and utility of facemask sampling in the detection of SARS-CoV-2 during an ongoing pandemic.

Easy access to screening for timely identification and isolation of infectious COVID-19 patients remains crucial in sustaining the international efforts to control COVID-19 spread. A major barrier limiting broad-based screening is the lack of a simple, rapid, and cost-effective COVID-19 testing method. We evaluated the feasibility and utility of facemask sampling in a cohort of 42 COVID-19-positive and 36 COVID-19-negative patients. We used a prototype of Steri-Strips™ (3 M) applied to the inner surface of looped surgical facemasks (Assure), which was worn by patients for a minimum wear time of 3 h, then removed and sent for SARS-CoV-2 PCR testing. Baseline demographics and symptomatology were also collected. Facemask sampling positivity was highest within the first 5 days of symptomatic presentation. Patients with nasopharyngeal and/or oropharyngeal swab SARS-CoV-2 PCR Ct values < 25.09 had SARS-CoV-2 detected on facemask sampling, while patients with Ct values ≥ 25.2 had no SARS-CoV-2 detected on facemask sampling. Facemask sampling can identify patients with COVID-19 during the early symptomatic phase or those with high viral loads, hence allowing timely identification and isolation of those with the highest transmission risk. Given the widespread use of facemasks, this method can potentially be easily applied to achieve broad-based, or even continuous, population screening.

Low incidence of venous thrombosis but high incidence of arterial thrombotic complications among critically ill COVID-19 patients in Singapore.

BACKGROUND: Arterial and venous thrombosis are reported to be common in critically ill COVID-19 patients. METHOD AND RESULTS: This is a national multicenter retrospective observational study involving all consecutive adult COVID-19 patients who required intensive care units (ICU) admission between 23 January 2020 and 30 April 2020 in Singapore. One hundred eleven patients were included and the venous and arterial thrombotic rates in ICU were 1.8% (n = 2) and 9.9% (n = 11), respectively. Major bleeding rate was 14.8% (n = 16). CONCLUSIONS: Critically ill COVID-19 patients in Singapore have lower venous thromboembolism but higher arterial thrombosis rates and bleeding manifestations than other reported cohorts.

Amplified parallel antigen rapid test for point-of-care salivary detection of SARS-CoV-2 with improved sensitivity.

In the ongoing COVID-19 pandemic, simple, rapid, point-of-care tests not requiring trained personnel for primary care testing are essential. Saliva-based antigen rapid tests (ARTs) can fulfil this need, but these tests require overnight-fasted samples; without which independent studies have demonstrated sensitivities of only 11.7 to 23.1%. Herein, we report an Amplified Parallel ART (AP-ART) with sensitivity above 90%, even with non-fasted samples. The virus was captured multimodally, using both anti-spike protein antibodies and Angiotensin Converting Enzyme 2 (ACE2) protein. It also featured two parallel flow channels. The first contained spike protein binding gold nanoparticles which produced a visible red line upon encountering the virus. The second contained signal amplifying nanoparticles that complex with the former and amplify the signal without any linker. Compared to existing dual gold amplification techniques, a limit of detection of one order of magnitude lower was achieved (0.0064 ng·mL-1). AP-ART performance in detecting SARS-CoV-2 in saliva of COVID-19 patients was investigated using a case-control study (139 participants enrolled and 162 saliva samples tested). Unlike commercially available ARTs, the sensitivity of AP-ART was maintained even when non-fasting saliva was used. Compared to the gold standard reverse transcription-polymerase chain reaction testing on nasopharyngeal samples, non-fasting saliva tested on AP-ART showed a sensitivity of 97.0% (95% CI: 84.7-99.8); without amplification, the sensitivity was 72.7% (95% CI: 83.7-94.8). Thus, AP-ART has the potential to be developed for point-of-care testing, which may be particularly important in resource-limited settings, and for early diagnosis to initiate newly approved therapies to reduce COVID-19 severity.

Coronavirus disease 2019 (COVID-19): an evidence map of medical literature.

BACKGROUND: Since the beginning of the COVID-19 outbreak in December 2019, a substantial body of COVID-19 medical literature has been generated. As of June 2020, gaps and longitudinal trends in the COVID-19 medical literature remain unidentified, despite potential benefits for research prioritisation and policy setting in both the COVID-19 pandemic and future large-scale public health crises. METHODS: In this paper, we searched PubMed and Embase for medical literature on COVID-19 between 1 January and 24 March 2020. We characterised the growth of the early COVID-19 medical literature using evidence maps and bibliometric analyses to elicit cross-sectional and longitudinal trends and systematically identify gaps. RESULTS: The early COVID-19 medical literature originated primarily from Asia and focused mainly on clinical features and diagnosis of the disease. Many areas of potential research remain underexplored, such as mental health, the use of novel technologies and artificial intelligence, pathophysiology of COVID-19 within different body systems, and indirect effects of COVID-19 on the care of non-COVID-19 patients. Few articles involved research collaboration at the international level (24.7%). The median submission-to-publication duration was 8 days (interquartile range: 4-16). CONCLUSIONS: Although in its early phase, COVID-19 research has generated a large volume of publications. However, there are still knowledge gaps yet to be filled and areas for improvement for the global research community. Our analysis of early COVID-19 research may be valuable in informing research prioritisation and policy planning both in the current COVID-19 pandemic and similar global health crises.

Characteristics of acute febrile illness and determinants of illness recovery among adults presenting to Singapore primary care clinics.

BACKGROUND: Undifferentiated acute febrile illness (AFI) is a common presentation among adults in primary care settings in Singapore but large gaps exist in the understanding of the characteristics of these patients. We studied clinical and epidemiological characteristics of AFI patients and factors associated with delayed recovery from AFI. METHODS: We performed a secondary data analysis using data from the Early DENgue infection and outcome (EDEN) study on 2046 adult patients presenting at 5 Singapore polyclinics between December 2007 and February 2013 with a history of fever (≥38 °C) for less than 72 h. We used an accelerated failure time model to investigate factors associated with delayed recovery from AFI. RESULTS: The mean age of patients was 36.6 years, 65 % were male, 51 % were of Chinese ethnicity, and 75 % lived in public housing. Median illness duration was 5 days (interquartile range, 3-7). In multivariable analysis, the unemployed and white collar workers had longer illness duration compared with blue collar workers (time ratio (TR), 1.10; 95 % confidence interval (CI), 1.03-1.17 and TR, 1.08; 95 % CI, 1.02-1.15, respectively). Patients with more symptoms at initial consultation had slower recovery (TR, 1.03 per additional symptom; 95 % CI, 1.02-1.03). Other clinical factors were also associated with longer duration of illness, including use of analgesics (TR, 1.21; 95 % CI, 1.15-1.28); use of cough medicines (TR, 1.14; 95 % CI, 1.08-1.20); use of antibiotics (TR, 1.14; 95 % CI, 1.07-1.21); and hospitalization (TR, 1.59; 95 % CI, 1.39-1.82). Compared to patients with normal WBC count at first consultation, those with low WBC count had slower recovery (TR, 1.14; 95 % CI, 1.07-1.21), while the reverse was observed among patients with high WBC count (TR, 0.94; 95 % CI, 0.88-1.00). CONCLUSIONS: Differences in illness duration among different types of employment may reflect differences in their underlying general health status. Early identification of factors delaying recovery could help triage management in a primary care setting. In-depth characterization of fever etiology in Singapore will improve surveillance and control activities.

Current status of silica-based nanoparticles as therapeutics and its potential as therapies against viruses.

In the past decade, interest in nanoparticles for clinical indications has been steadily gaining traction. Most recently, Lipid Nanoparticles (LNP) have been used successfully to construct the SARS-CoV-2 mRNA vaccines for rapid pandemic response. Similarly, silica is another nanomaterial which holds much potential to create nanomedicines against pathogens of interest. One major advantage of silica-based nanoparticles is its crystalline and highly ordered structure, which can be specifically tuned to achieve the desired properties needed for clinical applications. Increasingly, clinical research has shown the potential of silica nanoparticles not only as an antiviral, but also its ability as a delivery system for antiviral small molecules and vaccines against viruses. Silica has an excellent biosafety profile and has been tested in several early phase clinical trials since 2012, demonstrating good tolerability and minimal reported side effects. In this review, we discuss the clinical development of silica nanoparticles to date and identify the gaps and potential pitfalls in its path to clinical translation.

Use of analgesics/antipyretics in the management of symptoms associated with COVID-19 vaccination.

COVID-19 vaccines are effective and important to control the ongoing pandemic, but vaccine reactogenicity may contribute to poor uptake. Analgesics or antipyretic medications are often used to alleviate vaccine side effects, but their effect on immunogenicity remains uncertain. Few studies have assessed the effect of analgesics/antipyretics on vaccine immunogenicity and reactogenicity. Some studies revealed changes in certain immune response parameters post-vaccination when analgesics/antipyretics were used either prophylactically or therapeutically. Still, there is no evidence that these changes impact vaccine efficacy. Specific data on the impact of analgesic/antipyretic medications on immunogenicity of COVID-19 vaccines are limited. However, available data from clinical trials of licensed vaccines, along with recommendations from public health bodies around the world, should provide reassurance to both healthcare professionals and vaccine recipients that short-term use of analgesics/antipyretics at non-prescription doses is unlikely to affect vaccine-induced immunity.

SARS-CoV-2 breakthrough infection in vaccinees induces virus-specific nasal-resident CD8+ and CD4+ T cells of broad specificity.

Rapid recognition of SARS-CoV-2-infected cells by resident T cells in the upper airway might provide an important layer of protection against COVID-19. Whether parenteral SARS-CoV-2 vaccination or infection induces nasal-resident T cells specific for distinct SARS-CoV-2 proteins is unknown. We isolated T cells from the nasal mucosa of COVID-19 vaccinees who either experienced SARS-CoV-2 infection after vaccination (n = 34) or not (n = 16) and analyzed their phenotype, SARS-CoV-2 specificity, function, and persistence. Nasal-resident SARS-CoV-2-specific CD8+ and CD4+ T cells were detected almost exclusively in vaccinees who experienced SARS-CoV-2 breakthrough infection. Importantly, the Spike-specific T cells primed by vaccination did not suppress the induction of T cells specific for other SARS-CoV-2 proteins. The nasal-resident T cell responses persisted for ≥140 d, with minimal sign of waning. These data highlight the importance of viral nasal challenge in the formation of SARS-CoV-2-specific antiviral immunity at the site of primary infection and further define the immunological features of SARS-CoV-2 hybrid immunity.

Multifactorial influences underpinning a decision on COVID-19 vaccination among healthcare workers: a qualitative analysis.

COVID-19 vaccination in healthcare workers (HCW) is essential for improved patient safety and resilience of health systems. Despite growing body of literature on the perceptions of COVID vaccines in HCWs, existing studies tend to focus on reasons for 'refusing' the vaccines, using surveys almost exclusively. To gain a more nuanced understanding, we explored multifactorial influences underpinning a decision on vaccination and suggestions for decision support to improve vaccine uptake among HCWs in the early phase of vaccination rollout. Semi-structured interviews were undertaken with thirty-three HCWs in Singapore. Transcribed data was thematically analyzed. Decisions to accept vaccines were underpinned by a desire to protect patients primarily driven by a sense of professional integrity, collective responsibility to protect others, confidence in health authorities and a desire to return to a pre-pandemic way of life. However, there were prevailing concerns with respect to the vaccines, including long-term benefits, safety and efficacy, that hampered a decision. Inadequate information and social media representation of vaccination appeared to add to negative beliefs, impeding a decision to accept while low perceived susceptibility played a moderate role in the decision to delay or decline vaccination. Participants made valuable suggestions to bolster vaccination. Our findings support an approach to improving vaccine uptake in HCWs that features routine tracking and transparent updates on vaccination status, use of institutional platforms for sharing of experience, assuring contingency management plans and tailored communications to emphasize the duty of care and positive outlook associated with vaccination.

Favorable vaccine-induced SARS-CoV-2-specific T cell response profile in patients undergoing immune-modifying therapies.

BACKGROUNDPatients undergoing immune-modifying therapies demonstrate a reduced humoral response after COVID-19 vaccination, but we lack a proper evaluation of the effect of such therapies on vaccine-induced T cell responses.METHODSWe longitudinally characterized humoral and spike-specific T cell responses in patients with inflammatory bowel disease (IBD), who were on antimetabolite therapy (azathioprine or methotrexate), TNF inhibitors, and/or other biologic treatment (anti-integrin or anti-p40) for up to 6 months after completing 2-dose COVID-19 mRNA vaccination.RESULTSWe demonstrate that a spike-specific T cell response was not only induced in treated patients with IBD at levels similar to those of healthy individuals, but also sustained at higher magnitude for up to 6 months after vaccination, particularly in those treated with TNF inhibitor therapy. Furthermore, the spike-specific T cell response in these patients was mainly preserved against mutations present in SARS-CoV-2 B.1.1.529 (Omicron) and characterized by a Th1/IL-10 cytokine profile.CONCLUSIONDespite the humoral response defects, patients under immune-modifying therapies demonstrated a favorable profile of vaccine-induced T cell responses that might still provide a layer of COVID-19 protection.FUNDINGThis study was funded by the National Centre for Infectious Diseases (NCID) Catalyst Grant (FY2021ES) and the National Research Fund Competitive Research Programme (NRF-CRP25-2020-0003).

Differential immunogenicity of homologous versus heterologous boost in Ad26.COV2.S vaccine recipients.

BACKGROUND: Protection offered by coronavirus disease 2019 (COVID-19) vaccines wanes over time, requiring an evaluation of different boosting strategies to revert such a trend and enhance the quantity and quality of Spike-specific humoral and cellular immune responses. These immunological parameters in homologous or heterologous vaccination boosts have thus far been studied for mRNA and ChAdOx1 nCoV-19 vaccines, but knowledge on individuals who received a single dose of Ad26.COV2.S is lacking. METHODS: We studied Spike-specific humoral and cellular immunity in Ad26.COV2.S-vaccinated individuals (n = 55) who were either primed with Ad26.COV2.S only (n = 13) or were boosted with a homologous (Ad26.COV2.S, n = 28) or heterologous (BNT162b2, n = 14) second dose. We compared our findings with the results found in individuals vaccinated with a single (n = 16) or double (n = 44) dose of BNT162b2. FINDINGS: We observed that a strategy of heterologous vaccination enhanced the quantity and breadth of both Spike-specific humoral and cellular immunity in Ad26.COV2.S-vaccinated individuals. In contrast, the impact of the homologous boost was quantitatively minimal in Ad26.COV2.S-vaccinated individuals, and Spike-specific antibodies and T cells were narrowly focused to the S1 region. CONCLUSIONS: Despite the small sample size of the study and the lack of well-defined correlates of protection against COVID-19, the immunological features detected support the utilization of a heterologous vaccine boost in individuals who received Ad26.COV2.S vaccination. FUNDING: This study is partially supported by the Singapore Ministry of Health's National Medical Research Council under its COVID-19 Research Fund (COVID19RF3-0060, COVID19RF-001, and COVID19RF-008), The Medical College St. Bartholomew's Hospital Trustees - Pump Priming Fund for SMD COVID-19 Research.

Case report: Understanding the impact of persistent tissue-localization of SARS-CoV-2 on immune response activity via spatial transcriptomic analysis of two cancer patients with COVID-19 co-morbidity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected half a billion people, including vulnerable populations such as cancer patients. While increasing evidence supports the persistence of SARS-CoV-2 months after a negative nasopharyngeal swab test, the effects on long-term immune memory and cancer treatment are unclear. In this report, we examined post-COVID-19 tissue-localized immune responses in a hepatocellular carcinoma (HCC) patient and a colorectal cancer (CRC) patient. Using spatial whole-transcriptomic analysis, we demonstrated spatial profiles consistent with a lymphocyte-associated SARS-CoV-2 response (based on two public COVID-19 gene sets) in the tumors and adjacent normal tissues, despite intra-tumor heterogeneity. The use of RNAscope and multiplex immunohistochemistry revealed that the spatial localization of B cells was significantly associated with lymphocyte-associated SARS-CoV-2 responses within the spatial transcriptomic (ST) niches showing the highest levels of virus. Furthermore, single-cell RNA sequencing data obtained from previous (CRC) or new (HCC) ex vivo stimulation experiments showed that patient-specific SARS-CoV-2 memory B cells were the main contributors to this positive association. Finally, we evaluated the spatial associations between SARS-CoV-2-induced immunological effects and immunotherapy-related anti-tumor immune responses. Immuno-predictive scores (IMPRES) revealed consistent positive spatial correlations between T cells/cytotoxic lymphocytes and the predicted immune checkpoint blockade (ICB) response, particularly in the HCC tissues. However, the positive spatial correlation between B cells and IMPRES score was restricted to the high-virus ST niche. In addition, tumor immune dysfunction and exclusion (TIDE) analysis revealed marked T cell dysfunction and inflammation, alongside low T cell exclusion and M2 tumor-associated macrophage infiltration. Our results provide in situ evidence of SARS-CoV-2-generated persistent immunological memory, which could not only provide tissue protection against reinfection but may also modulate the tumor microenvironment, favoring ICB responsiveness. As the number of cancer patients with COVID-19 comorbidity continues to rise, improved understanding of the long-term immune response induced by SARS-CoV-2 and its impact on cancer treatment is much needed.

Clinical and laboratory features of hypercoagulability in COVID-19 and other respiratory viral infections amongst predominantly younger adults with few comorbidities.

COVID-19 caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and other respiratory viral (non-CoV-2-RV) infections are associated with thrombotic complications. The differences in prothrombotic potential between SARS-CoV-2 and non-CoV-2-RV have not been well characterised. We compared the thrombotic rates between these two groups of patients directly and further delved into their coagulation profiles. In this single-center, retrospective cohort study, all consecutive COVID-19 and non-CoV-2-RV patients admitted between January 15th and April 10th 2020 were included. Coagulation parameters studied were prothrombin time and activated partial thromboplastin time and its associated clot waveform analysis (CWA) parameter, min1, min2 and max2. In the COVID-19 (n = 181) group there were two (1.0 event/1000-hospital-days) myocardial infarction events while one (1.8 event/1000-hospital-day) was reported in the non-CoV-2-RV (n = 165) group. These events occurred in patients who were severely ill. There were no venous thrombotic events. Coagulation parameters did not differ throughout the course of mild COVID-19. However, CWA parameters were significantly higher in severe COVID-19 compared with mild disease, suggesting hypercoagulability (min1: 6.48%/s vs 5.05%/s, P < 0.001; min2: 0.92%/s2 vs 0.74%/s2, P = 0.033). In conclusion, the thrombotic rates were low and did not differ between COVID-19 and non-CoV-2-RV patients. The hypercoagulability in COVID-19 is a highly dynamic process with the highest risk occurring when patients were most severely ill. Such changes in haemostasis could be detected by CWA. In our population, a more individualized thromboprophylaxis approach, considering clinical and laboratory factors, is preferred over universal pharmacological thromboprophylaxis for all hospitalized COVID-19 patients and such personalized approach warrants further research.