ABSTRACT
Immunization with mRNA or viral vectors encoding spike with diproline substitutions (S-2P) has provided protective immunity against severe COVID-19 disease. How immunization with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike elicits neutralizing antibodies (nAbs) against difficult-to-neutralize variants of concern (VOCs) remains an area of great interest. Here, we compare immunization of macaques with mRNA vaccines expressing ancestral spike either including or lacking diproline substitutions, and show the diproline substitutions were not required for protection against SARS-CoV-2 challenge or induction of broadly neutralizing B cell lineages. One group of nAbs elicited by the ancestral spike lacking diproline substitutions targeted the outer face of the receptor binding domain (RBD), neutralized all tested SARS-CoV-2 VOCs including Omicron XBB.1.5, but lacked cross-Sarbecovirus neutralization. Structural analysis showed that the macaque broad SARS-CoV-2 VOC nAbs bound to the same epitope as a human broad SARS-CoV-2 VOC nAb, DH1193. Vaccine-induced antibodies that targeted the RBD inner face neutralized multiple Sarbecoviruses, protected mice from bat CoV RsSHC014 challenge, but lacked Omicron variant neutralization. Thus, ancestral SARS-CoV-2 spike lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to distinct RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs.
Subject(s)
Severe Acute Respiratory Syndrome , COVID-19ABSTRACT
Age is among the strongest risk factors for severe outcomes from SARS-CoV-2 infection. We sought to evaluate associations between age and both mucosal and systemic host responses to SARS-CoV-2 infection. We profiled the upper respiratory tract (URT) and peripheral blood transcriptomes of 201 participants (age range of 1 week to 83 years), including 137 non-hospitalized individuals with mild SARS-CoV-2 infection and 64 uninfected individuals. Among uninfected children and adolescents, young age was associated with upregulation of innate and adaptive immune pathways within the URT, suggesting that young children are primed to mount robust mucosal immune responses to exogeneous respiratory pathogens. SARS-CoV-2 infection was associated with broad induction of innate and adaptive immune responses within the URT of children and adolescents. Peripheral blood responses among SARS-CoV-2-infected children and adolescents were dominated by interferon pathways, while upregulation of myeloid activation, inflammatory, and coagulation pathways was observed only in adults. Systemic symptoms among SARS-CoV-2-infected subjects were associated with blunted innate and adaptive immune responses in the URT and upregulation of many of these same pathways within peripheral blood. Finally, within individuals, robust URT immune responses were correlated with decreased peripheral immune activation, suggesting that effective immune responses in the URT may promote local viral control and limit systemic immune activation and symptoms. These findings demonstrate that there are differences in immune responses to SARS-CoV-2 across the lifespan, including between young children and adolescents, and suggest that these varied host responses contribute to observed differences in the clinical presentation of SARS-CoV-2 infection by age.
Subject(s)
COVID-19 , Severe Acute Respiratory SyndromeABSTRACT
Real-time, reverse transcriptase PCR assays are a pervasive technology used for diagnosis of SARS-CoV-2 infection. These assays produce a cycle threshold value (Ct) corresponding to the first amplification cycle in which reliable amplification is detected. Such Ct values have been used by clinicians and in public health settings to guide treatment, monitor disease progression, assess prognosis, and inform isolation practices. To understanding the risk of reporting out uncalibrated Ct values and potential for instead reporting out calibrated viral load values, we performed a multi-institutional study to benchmark major clinical platforms against a calibrated standard. We found that for any given Ct value, corresponding viral loads varied up to 1000-fold among the different tests. In contrast, when these different assays were calibrated against a common standard and then used to test unknown de-identified specimens at several dilutions, viral load values showed high precision between methods (standard deviation and range of 0.36 and 1.1 log10 genome copies) and high accuracy compared with droplet digital PCR (ddPCR) determinations (difference between mean CDC N2 and Sarbeco E ddPCR determinations and mean determinations by calibrated RT-PCR assays examined in our study of 0.044 log10 genome copies). We, therefore, find strong support for calibration of SARS-CoV-2 RT-PCR tests to allow conversion of cycle thresholds to accurate and precise viral load values that are reproducible across major clinical systems. Implementation of calibrated assays will provide more reliable information for clinical decision making and allow more rigorous interpretation of SARS-CoV-2 laboratory data in clinical and laboratory investigation.
Subject(s)
COVID-19ABSTRACT
Coronavirus vaccines that are highly effective against SARS-CoV-2 variants are needed to control the current pandemic. We previously reported a receptor-binding domain (RBD) sortase A-conjugated ferritin nanoparticle (RBD-scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected monkeys from SARS-CoV-2 WA-1 infection. Here, we demonstrate SARS-CoV-2 RBD-scNP immunization induces potent neutralizing antibodies against all eight SARS-CoV-2 variants tested including the Beta, Delta, and Omicron variants in non-human primates (NHPs). The Omicron variant was neutralized by RBD-scNP-induced serum antibodies with a mean of 4.3-fold reduction of ID50 titers compared to SARS-CoV-2 D614G. Immunization with RBD-scNPs protected NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protected mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect NHPs and mice from multiple different SARS-related viruses. Such a vaccine could provide the needed immunity to slow the spread of and reduce disease caused by SARS-CoV-2 variants such as Delta and Omicron.
Subject(s)
Severe Acute Respiratory SyndromeABSTRACT
Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.
Subject(s)
Coronavirus Infections , Severe Acute Respiratory SyndromeABSTRACT
SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19, making them a focus of vaccine design. A safety concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated potent NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike protein from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV-1 infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of antibody binding. Select RBD NAbs also demonstrated Fc receptor-{gamma} (Fc{gamma}R)-mediated enhancement of virus infection in vitro, while five non-neutralizing NTD antibodies mediated Fc{gamma}R-independent in vitro infection enhancement. However, both in vitro neutralizing and infection-enhancing RBD or infection-enhancing NTD antibodies protected from SARS-CoV-2 challenge in non-human primates and mice. One of 30 monkeys infused with enhancing antibodies had lung pathology and bronchoalveolar lavage cytokine evidence suggestive of enhanced disease. Thus, these in vitro assessments of enhanced antibody-mediated infection do not necessarily indicate biologically relevant in vivo infection enhancement.
Subject(s)
Severe Acute Respiratory Syndrome , Tumor Virus Infections , COVID-19ABSTRACT
Host-virus protein-protein interaction is the key component of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lifecycle. We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Functional characterization and further validation of these interactions elucidated how distinct SARS-CoV-2 viral proteins participate in its lifecycle, and discovered potential drug targets to the treatment of COVID-19. The interactomes of two key SARS-CoV-2 encoded viral proteins, NSP1 and N protein, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of coronavirus disease-2019 provides valuable resources for understanding and treating this disease.
Subject(s)
Coronavirus Infections , COVID-19ABSTRACT
The ongoing coronavirus disease 2019 (COVID-19) pandemic has become a serious global threat. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for this pandemic has imposed a severe burden on the medical settings. The spike (S) protein of SARS-CoV-2 is an important structural protein playing a key role in the viral entry. This protein is responsible for the receptor recognition and cell membrane fusion process. The recent reports of the appearance and spread of new SARS-CoV-2 strain has raised alarms. It was reported that this new variant containing the prominent active site mutation in the RBD (N501Y) was rapidly spreading within the population. The reported N501Y mutation within the spike's essential part, known as the receptor-binding domain has raised several questions. Here in this study we have tried to explore the effect of N501Y mutation within the spike protein using several in silico approaches
Subject(s)
Coronavirus Infections , Severe Acute Respiratory Syndrome , COVID-19ABSTRACT
COVID-19, caused by SARS-CoV-2, was first reported in China in 2019 and has transmitted rapidly around the world, currently responsible for 83 million reported cases and over 1.8 million deaths. The mode of transmission is believed principally to be airborne exposure to respiratory droplets from symptomatic and asymptomatic patients but there is also a risk of the droplets contaminating fomites such as touch surfaces including door handles, stair rails etc, leading to hand pick up and transfer to eyes, nose and mouth. We have previously shown that human coronavirus 229E survives for more than 5 days on inanimate surfaces and another laboratory reproduced this for SARS-CoV-2 this year. However, we showed rapid inactivation of Hu-CoV-229E within 10 minutes on different copper surfaces while the other laboratory indicated this took 4 hours for SARS-CoV-2. So why the difference? We have repeated our work with SARS-CoV-2 and can confirm that this coronavirus can be inactivated on copper surfaces in as little as 1 minute. We discuss why the 4 hour result may be technically flawed.
Subject(s)
COVID-19ABSTRACT
SARS-CoV-2 infection of the respiratory system can evolve to a multi-system disease. Excessive levels of proinflammatory cytokines, known as a "cytokine storm" are associated with high mortality rates especially in the elderly and in patients with age-related morbidities. Senescent cells, characterized by secretion of such cytokines (Senescence Associated Secretory Phenotype - SASP), are known to occur in this context as well as upon a variety of stressogenic insults. Applying both: i) a novel "in house" antibody against the spike protein of SARS-CoV-2 and ii) a unique senescence detecting methodology, we identified for the first time in lung tissue from COVID-19 patients alveolar cells acquiring senescent features harboring also SARS-CoV-2. Moreover, using the same detection workflow we demonstrated the inflammatory properties of these cells. Our findings justify the application of senotherapeutics for the treatment or prevention of COVID-19 patients.
Subject(s)
Adenocarcinoma, Bronchiolo-Alveolar , COVID-19ABSTRACT
BACKGROUNDChildren with SARS-CoV-2 infection typically have mild symptoms that do not require medical attention, leaving a gap in our understanding of the spectrum of illnesses that the virus causes in children. METHODSWe conducted a prospective cohort study of children and adolescents (<21 years of age) with a SARS-CoV-2-infected close contact. We collected nasopharyngeal or nasal swabs at enrollment and tested for SARS-CoV-2 using a real-time PCR assay. RESULTSOf 382 children, 289 (76%) were SARS-CoV-2-infected. SARS-CoV-2-infected children were more likely to be Hispanic (p<0.0001), less likely to have a history of asthma (p=0.009), and more likely to have an infected sibling contact (p=0.0007) than uninfected children. Children ages 6-13 years were frequently asymptomatic (38%) and had respiratory symptoms less often than younger children (30% vs. 49%; p=0.008) or adolescents (30% vs. 59%; p<0.0001). Compared to children ages 6-13 years, adolescents more frequently reported influenza-like (61% vs. 39%; p=0.002), gastrointestinal (26% vs. 9%; p=0.003), and sensory symptoms (43% vs. 9%; p<0.0001), and had more prolonged illnesses [median (IQR) duration: 7 (4, 12) vs. 4 (3, 8) days; p=0.004]. Despite the age-related variability in symptoms, we found no differences in nasopharyngeal viral load by age or between symptomatic and asymptomatic children. CONCLUSIONSHispanic ethnicity and an infected sibling close contact are associated with increased SARS-CoV-2 infection risk among children, while a history of asthma is associated with decreased risk. Age-related differences in the clinical manifestations of SARS-CoV-2 infection must be considered when evaluating children for COVID-19 and in developing screening strategies for schools and childcare settings.