Studies have found different waning rates of neutralising antibodies compared with binding antibodies against SARS-CoV-2. The impact of neutralising antibody waning rate at the individual patient level on the longevity of immunity remains unknown. We aimed to investigate the peak levels and dynamics of neutralising antibody waning and IgG avidity maturation over time, and correlate this with clinical parameters, cytokines, and T-cell responses.We did a longitudinal study of patients who had recovered from COVID-19 up to day 180 post-symptom onset by monitoring changes in neutralising antibody levels using a previously validated surrogate virus neutralisation test. Changes in antibody avidities and other immune markers at different convalescent stages were determined and correlated with clinical features. Using a machine learning algorithm, temporal change in neutralising antibody levels was classified into five groups and used to predict the longevity of neutralising antibody-mediated immunity.We approached 517 patients for participation in the study, of whom 288 consented for outpatient follow-up and collection of serial blood samples. 164 patients were followed up and had adequate blood samples collected for analysis, with a total of 546 serum samples collected, including 128 blood samples taken up to 180 days post-symptom onset. We identified five distinctive patterns of neutralising antibody dynamics as follows: negative, individuals who did not, at our intervals of sampling, develop neutralising antibodies at the 30% inhibition level (19 [12%] of 164 patients); rapid waning, individuals who had varying levels of neutralising antibodies from around 20 days after symptom onset, but seroreverted in less than 180 days (44 [27%] of 164 patients); slow waning, individuals who remained neutralising antibody-positive at 180 days post-symptom onset (46 [28%] of 164 patients); persistent, although with varying peak neutralising antibody levels, these individuals had minimal neutralising antibody decay (52 [32%] of 164 patients); and delayed response, a small group that showed an unexpected increase of neutralising antibodies during late convalescence (at 90 or 180 days after symptom onset; three [2%] of 164 patients). Persistence of neutralising antibodies was associated with disease severity and sustained level of pro-inflammatory cytokines, chemokines, and growth factors. By contrast, T-cell responses were similar among the different neutralising antibody dynamics groups. On the basis of the different decay dynamics, we established a prediction algorithm that revealed a wide range of neutralising antibody longevity, varying from around 40 days to many decades.Neutralising antibody response dynamics in patients who have recovered from COVID-19 vary greatly, and prediction of immune longevity can only be accurately determined at the individual level. Our findings emphasise the importance of public health and social measures in the ongoing pandemic outbreak response, and might have implications for longevity of immunity after vaccination.National Medical Research Council, Biomedical Research Council, and A*STAR, Singapore.
Summary Understanding mucosal antibody responses from SARS-CoV-2 infection and/or vaccination is crucial to develop strategies for longer term immunity, especially against emerging viral variants. We profiled serial paired mucosal and plasma antibodies from: COVID-19 vaccinated only vaccinees (vaccinated, uninfected), COVID-19 recovered vaccinees (convalescent, vaccinated) and individuals with breakthrough Delta or Omicron BA.2 infections (vaccinated, infected). Saliva from COVID-19 recovered vaccinees displayed improved antibody neutralizing activity, FcγR engagement and IgA compared to COVID-19 uninfected vaccinees. Furthermore, repeated mRNA vaccination boosted SARS-CoV-2-specific IgG2 and IgG4 responses in both mucosa biofluids (saliva and tears) and plasma. IgG, but not IgA, responses to breakthrough COVID-19 variants were dampened and narrowed by increased pre-existing vaccine-induced immunity to the ancestral strain. Salivary antibodies delayed initiation of boosting following breakthrough COVID-19 infection, especially Omicron BA.2, however, rose rapidly thereafter. Our data highlight how pre-existing immunity shapes mucosal SARS-CoV-2-specific antibody responses and has implications for long-term protection from COVID-19.
Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide, causing severe infections and death with hitherto no proven therapy.Methods: This was a prospective observational study of 100 patients with virologically confirmed 2019 novel coronavirus disease (COVID-19) admitted to public hospitals in Singapore from 22 January to 6 March 2020. Enrolled patients contributed clinical data from medical records, respiratory samples for SARS-CoV-2 polymerase chain reaction (PCR) and viral culture, serial serum samples for IgG and IgM SARS-CoV-2 serology and plasma samples for immune-profiling.Findings: Of the 100 patients, mean age was 46 years (95% confidence interval [CI] 43-49), males comprised 56 (56%) and 38 (38%) had comorbidities. Median time from symptom onset to hospital admission was 5·3 days (inter-quartile range (IQR) 1·3-8). Fifty-seven (57%) developed viral pneumonia, of whom 20 (20%) required supplemental oxygen including 12 (12%) invasive mechanical ventilation. Viral culture from respiratory samples was positive for 14 of 73 patients (19%). PCR cycle threshold value ≤30 predicted viral isolation by culture or next-generation sequencing (p<0·0001). Average duration of viral shedding by PCR was 16·7 days (95% CI 15·2-18·3) which did not differ significantly by disease severity or treatment with lopinavir-ritonavir. Seroconversion occurred at a median of 12·5 days (IQR 9-18) for IgM and 15·0 days (IQR 12-20) for IgG; 49/51 patients (96.1%) sampled at day 14 or later seroconverted. Severe infections were associated with earlier seroconversion and higher peak IgM and IgG levels. Levels of IP-10, HGF, IL-6, MCP-1, MIP-1α, IL-12p70, IL-18, VEGF-A, PDGF-BB and IL-1RA significantly correlated with disease severity.Interpretation: Our findings of virus viability have implications on infectivity and infection control. The timing of seroconversion may guide use of rapid serological assays and convalescent plasma collection from donors. The identified overactive proinflammatory immune signatures suggest targets for host-directed immunotherapy which should be evaluated in randomised controlled trials.Funding Statement: National Medical Research Council COVID research fund (COVID19RF-001). This work is supported in part by the Singapore NMRC grants STPRG-FY19-001 and COVID19RF-003, and Singapore NRF grant NRF2016NRF-NSFC002-013 and by a core fund by the Singapore Immunology Network, Agency for Science Technology and Research (A*STAR).Declaration of Interests: BY reports personal fees from Roche and Sanofi, outside the submitted work. All other authors: no interest declared.Ethics Approval Statement: Written informed consent was obtained from all study participants (study protocol at all sites approved by the National Healthcare Group Domain Specific Review Board, Study Reference 2012/00917; additional study protocol at Singapore General Hospital approved by the SingHealth Centralised Institutional Review Board, Study Reference 2018/3045).
ABSTRACT 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 (T H 1) 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 T H 1-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.
The novel coronavirus SARS-CoV-2 is the causative agent of Coronavirus Disease 2019 (COVID-19), a global healthcare and economic catastrophe. Understanding of the host immune response to SARS-CoV-2 is still in its infancy. A 382-nt deletion strain lacking ORF8 (Δ382 herein) was isolated in Singapore in March 2020. Infection with Δ382 was associated with less severe disease in patients, compared to infection with wild-type SARS-CoV-2. Here, we established Nasal Epithelial cells (NECs) differentiated from healthy nasal-tissue derived stem cells as a suitable model for the ex-vivo study of SARS-CoV-2 mediated pathogenesis. Infection of NECs with either SARS-CoV-2 or Δ382 resulted in virus particles released exclusively from the apical side, with similar replication kinetics. Screening of a panel of 49 cytokines for basolateral secretion from infected NECs identified CXCL10 as the only cytokine significantly induced upon infection, at comparable levels in both wild-type and Δ382 infected cells. Transcriptome analysis revealed the temporal up-regulation of distinct gene subsets during infection, with anti-viral signaling pathways only detected at late time-points (72 hours post-infection, hpi). This immune response to SARS-CoV-2 was significantly attenuated when compared to infection with an influenza strain, H3N2, which elicited an inflammatory response within 8 hpi, and a greater magnitude of anti-viral gene up-regulation at late time-points. Remarkably, Δ382 induced a host transcriptional response nearly identical to that of wild-type SARS-CoV-2 at every post-infection time-point examined. In accordance with previous results, Δ382 infected cells showed an absence of transcripts mapping to ORF8, and conserved expression of other SARS-CoV-2 genes. Our findings shed light on the airway epithelial response to SARS-CoV-2 infection, and demonstrate a non-essential role for ORF8 in modulating host gene expression and cytokine production from infected cells.
Abstract The SARS-CoV-2 B.1.1.529 lineage, Omicron variant, was first detected in November 2021 and carries 32 amino acid mutations in the spike protein (15 in RBD) and exhibits significant escape of neutralizing antibodies targeting the parental SARS-CoV-2 virus. Here, we performed a high-resolution multiplex (16-plex) surrogate virus neutralization assay covering all major SARS-CoV-2 variants and pre-emergent ACE2-binding sarbecoviruses against 20 different human serum panels from infected, vaccinated and hybrid immune individuals which had vaccine-breakthrough infections or infection followed by vaccination. Among all sarbecoviruses tested, we observed 1.1 to 4.7-, 2.3 to 10.3- and 0.7 to 33.3-fold reduction in neutralization activities to SARS-CoV-2 Beta, Omicron and SARS-CoV-1, respectively. Among the SARS-CoV-2 related sarbecoviruses, it is found that the genetically more distant bat RaTG13 and pangolin GX-P5L sarbecoviruses had less neutralization escape than Omicron. Our data suggest that the SARS-CoV-2 variants emerged from the changed immune landscape of human populations are more potent in escaping neutralizing antibodies, from infection or vaccination, than pre-emergent sarbecoviruses naturally evolved in animal populations with no or less immune selection pressure.
The recent emergence and spread of zoonotic viruses highlights that animal-sourced viruses are the biggest threat to global public health. Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an HKU2-related bat coronavirus that was spilled over from Rhinolophus bats to swine, causing large-scale outbreaks of severe diarrhea disease in piglets in China. Unlike other porcine coronaviruses, SADS-CoV possesses broad species tissue tropism, including primary human cells, implying a significant risk of cross-species spillover. To explore host dependency factors for SADS-CoV as therapeutic targets, we employed genome-wide CRISPR knockout library screening in HeLa cells. Consistent with two independent screens, we identified the zinc finger DHHC-type palmitoyltransferase 17 (ZDHHC17 or ZD17) as an important host factor for SADS-CoV infection. Through truncation mutagenesis, we demonstrated that the DHHC domain of ZD17 that is involved in palmitoylation is important for SADS-CoV infection. Mechanistic studies revealed that ZD17 is required for SADS-CoV genomic RNA replication. Treatment of infected cells with the palmitoylation inhibitor 2-bromopalmitate (2-BP) significantly suppressed SADS-CoV infection. Our findings provide insight on SADS-CoV-host interactions and a potential therapeutic application. IMPORTANCE The recent emergence of deadly zoonotic viral diseases, including Ebola virus and SARS-CoV-2, emphasizes the importance of pandemic preparedness for the animal-sourced viruses with potential risk of animal-to-human spillover. Over the last 2 decades, three significant coronaviruses of bat origin, SARS-CoV, MERS-CoV, and SARS-CoV-2, have caused millions of deaths with significant economy and public health impacts. Lack of effective therapeutics against these coronaviruses was one of the contributing factors to such losses. Although SADS-CoV, another coronavirus of bat origin, was only known to cause fatal diarrhea disease in piglets, the ability to infect cells derived from multiple species, including human, highlights the potential risk of animal-to-human spillover. As part of our effort in pandemic preparedness, we explore SADS-CoV host dependency factors as targets for host-directed therapeutic development and found zinc finger DHHC-type palmitoyltransferase 17 is a promising drug target against SADS-CoV replication. We also demonstrated that a palmitoylation inhibitor, 2-bromopalmitate (2-BP), can be used as an inhibitor for SADS-CoV treatment.
W ith a population exceeding 630 million (8% of the global population), the ten member states of the Association of Southeast Asian Nations (ASEAN) accounted for approximately 61 million (7.9%) of global COVID-19 cases and 808 166 (11.6%) of deaths, with case fatality rate (CFR) of 1.3% by the end of 2023 [1].The actual figures are certain to be at least several-fold higher, with estimates suggesting an excess mortality of 1.2 million in the first two years of the pandemic.Despite the World Health Organization's (WHO) 4 May 2023 declaration that coronavirus disease 2019 (COVID-19) is no longer a public health emergency, it remains a global threat.The global response to the COVID-19 pandemic has highlighted the importance of collaborative networks within ASEAN, particularly in scientific information, technology and research exchange, resource mobilisation, and capacity building.While individual ASEAN country implemented responses with varying degrees of success, a unified and cohesive regional approach is crucial for
Sarbecoviruses are a subgenus of Coronaviridae that mostly infect bats with known potential to infect humans (SARS-CoV and SARS-CoV-2). Populations in Southeast Asia, where these viruses are most likely to emerge, have been undersurveyed to date.
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