Spike Protein Research Articles

Evaluation of Cellular Immune Responses After mRNA-1273 Vaccination in Children 6 Months to 11 Years of Age.

Cell-mediated immunity (CMI) may help protect against emerging SARS-CoV-2 variants that are less susceptible to neutralizing antibodies. We present CMI data after the mRNA-1273 primary series in a subset of participants aged 6 months to 11 years from the phase 2/3 KidCOVE trial. T-cell responses were assessed after 2 doses of mRNA-1273 (6 months-5 years: 25 μg; 6-11 years: 50 μg) or placebo administered 28 days apart. Magnitude, phenotype, and percentage of ancestral SARS-CoV-2 spike (S) protein T-cell responses to pooled peptides were assessed by intracellular cytokine staining and polyfunctionality analyses. A total of 68 children aged 6 months to 11 years received either the 2-dose mRNA-1273 primary series or placebo (51:17, respectively) at 28-day interval. mRNA-1273 induced S protein-specific CD4+ T-cell responses exhibiting a type 1 T helper (Th1)-biased profile at Day 43 and Day 209 compared with placebo. S-protein-specific CD8+ T-cell responses were less frequently detected in children <5 years and undetectable in those <2 years. Compared with placebo, mRNA-1273 induced higher frequencies of S-specific polyfunctional CD4+ T cells at Day 43; frequencies declined but remained detectable at Day 209. Correlation between Th1 CD4+ responses and neutralizing antibodies was observed across age groups following mRNA-1273 vaccination. The 2-dose mRNA-1273 primary series elicited robust and durable (≥6 months) Th1-biased CD4+ T-cell responses in children aged 6 months to 11 years. CD8+ T-cell responses varied by age.Trial registration number and URL NCT04796896 (https://clinicaltrials.gov/study/NCT04796896).

Whole-body visualization of SARS-CoV-2 biodistribution in vivo by immunoPET imaging in non-human primates

The COVID-19 pandemic has caused at least 780 million cases globally. While available treatments and vaccines have reduced the mortality rate, spread and evolution of the virus are ongoing processes. Despite extensive research, the long-term impact of SARS-CoV-2 infection is still poorly understood and requires further investigation. Routine analysis provides limited access to the tissues of patients, necessitating alternative approaches to investigate viral dissemination in the organism. We address this issue by implementing a whole-body in vivo imaging strategy to longitudinally assess the biodistribution of SARS-CoV-2. We demonstrate in a COVID-19 non-human primate model that a single injection of radiolabeled [89Zr]COVA1-27-DFO human monoclonal antibody targeting a preserved epitope of the SARS-CoV-2 spike protein allows longitudinal tracking of the virus by positron emission tomography with computed tomography (PET/CT). Convalescent animals exhibit a persistent [89Zr]COVA1-27-DFO PET signal in the lungs, as well as in the brain, three months following infection. This imaging approach also allows viral detection in various organs, including the airways and kidneys, of exposed animals during the acute infection phase. Overall, the technology we developed offers a comprehensive assessment of SARS-CoV-2 distribution in vivo and provides a promising approach for the non-invasive study of long-COVID pathophysiology.

Computationally designed multi-epitope vaccine construct targeting the SARS-CoV-2 spike protein elicits robust immune responses in silico

Our research is driven by the need to design an advanced multi-epitope vaccine construct (MEVC) using the S-protein of SARS-CoV-2 to combat the emergence of new variants. Through rigorous computational screening, we have identified linear and discontinuous B-cell epitopes, CD8 + and CD4 + T-cell epitopes, ensuring extensive MEVC coverage across 90.03% of the global population. The MEVC, featuring four CD4 + and four CD8 + T-cell epitopes connected linearly with two adjuvant proteins on both ends, has been carefully designed to elicit robust immune response. Our in-silico analysis has confirmed the construct’s antigenicity, non-allergenicity, and non-toxicity with optimized codon sequences for enhanced expression in E. coli K12. Furthermore, molecular docking and dynamics analyses have demonstrated its strong binding affinity with TLR-3 and TLR 4, and in-silico immune simulation yielded promising results on heightened B-cell and T-cell-mediated immunity. However, wet lab experiments are essential to validate computational findings to revolutionize the development of vaccines against SARS-CoV-2.

Internalization of affinity tags enables the purification of secreted Chlamydomonas proteins

There is great interest in establishing microalgae as new platforms for the sustainable production of high-value products such as recombinant proteins. Many human therapeutic proteins must be glycosylated, which requires their passage through the secretory pathway into the culture medium. While the low complexity of proteins in the culture medium should facilitate affinity purification of secreted recombinant proteins, this has proven challenging for proteins secreted by the unicellular green alga Chlamydomonas reinhardtii. In Leishmania tarentulae, we observed that C-terminally exposed affinity tags are frequently truncated, presumably due to proteolytic activity. We wondered whether this might also occur in Chlamydomonas and contribute to the difficulties in affinity purification of secreted proteins in this alga. Using the methionine-rich 2S albumin from Bertholletia excelsa and the ectodomain of the SARS-CoV-2 spike protein produced and secreted in Chlamydomonas, we demonstrate that they can be efficiently affinity-purified from the culture medium by Ni-NTA chromatography when the 8xHis affinity tag is internalized. This finding represents an important step towards further development of Chlamydomonas as a host for the sustainable production of high-value recombinant proteins.

Spike Protein–Fibrinogen Interaction: A Novel Immune Evasion Strategy of SARS-CoV-2?

Spike Protein–Fibrinogen Interaction: A Novel Immune Evasion Strategy of SARS-CoV-2?

Clinical and molecular landscape of prolonged SARS-CoV-2 infection with resistance to remdesivir in immunocompromised patients

Abstract Patients with hematologic diseases have experienced COVID-19 with prolonged, progressive course. Here we present clinical, pathological, and virological analyses of three cases of prolonged COVID-19 among patients undergoing treatment for B-cell lymphoma. These patients had all been treated with anti-CD20 antibody and bendamustine. Despite various antiviral treatments, high SARS-CoV-2 levels persisted for more than 4 weeks, and two of them succumbed to COVID-19. Autopsy showed bronchopneumonia, interstitial pneumonia, alveolar hemorrhage, and fibrosis. Overlapping CMV, fungal and/or bacterial infections were also confirmed. Sequencing of SARS-CoV-2 showed accumulation of mutations and changes in variant allele frequencies over time. NSP12 mutations V792I and M794I appeared independently in two cases as COVID-19 progressed. In vitro drug susceptibility analysis and animal experiment using recombinant SARS-CoV-2 demonstrated that each mutation, V792 and M794I, was independently responsible for remdesivir resistance and attenuated pathogenicity. E340A, E340D and F342INS mutations in the spike protein were found in one case, which may account for the sotrovimab resistance. Analysis of autopsy specimens indicated heterogeneous distribution of these mutations. In summary, we demonstrated temporal and spatial diversity in SARS-CoV-2 that evolved resistance to various antiviral agents in malignant lymphoma patients under immunodeficient conditions caused by certain types of immunochemotherapies. Strategies may be necessary to prevent acquisition of drug resistance and improve outcome, such as selection of appropriate treatment strategies for lymphoma considering patients’ immune status and institution of early intensive antiviral therapy.

Time course and determinants of the antibody response to SARS-CoV-2 in Costa Rica: the RESPIRA study

BackgroundAntibodies to SARS-CoV-2 are essential for protection or reduction in severity of subsequent disease. We studied antibody responses to spike protein receptor-binding domain (S1-RBD) and nucleocapsid (N) in a population-based sample of COVID-19 cases in Costa Rica.MethodsAs part of the RESPIRA study, we selected an age-stratified random sample of PCR-confirmed COVID-19 cases diagnosed from March 2020 to July 2021. Antibodies were determined with multiplex serology in 794 unvaccinated subjects diagnosed 3 days to 17 months before recruitment to investigate immune response to natural infection. In addition, neutralizing antibodies were determined in 136 randomly selected participants. We estimated antibody positivity and GMTs by time since diagnosis and explored determinants using multivariate regression.ResultsMost participants tested 15–29 days after PCR diagnosis were seropositive for N (90%) and S1-RBD antibodies (96%) and had the highest GMTs for both antibodies. Only 42% of subjects tested one year after infection were seropositive for N antibodies, compared to 97% for S1-RBD. GMTs for neutralizing antibodies peaked 15–89 days after infection and declined but remained positive for 95% of subjects thereafter. In multivariate models, antibodies were significantly higher among men and increased with age and severity of the clinical presentation. The correlation of multiplex and neutralizing antibodies was high (0.72 [95% CI = 0.63–0.79]) and stronger among women.ConclusionsA robust immune response against N and S1-RBD is elicited by COVID-19 a few days after infection. While S1-RBD antibodies are present after > 1 year, N antibodies decline significantly. Antibody levels are higher in men and increase with age and severity of disease. The different immune response patterns by sex warrant further investigation.Trial registrationRESPIRA Study ClinicalTrials.gov ID: NCT04537338 (3 September 2020).

Probiotics influencing response of antibodies over time in seniors after COVID-19 vaccine (PIRATES-COV): a randomised controlled trial protocol

IntroductionThe elderly are particularly vulnerable to morbidity and mortality from COVID-19, the disease caused by the SARS-CoV-2. Approximately 20% of the elderly showed no antibodies 3–5 months post-second dose of...

Cnidii fructus and Sophorae Flavescentis Radix polysaccharides inhibit SARS-CoV-2 entry by interfering with Spike protein-mediated membrane fusion.

Cnidii fructus and Sophorae Flavescentis Radix polysaccharides inhibit SARS-CoV-2 entry by interfering with Spike protein-mediated membrane fusion.

Investigating the mechanisms of ethanol-induced disruption of COVID-19 lipid bilayers through molecular dynamics simulations.

The COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, began in December 2019 in Wuhan, China. To mitigate the spread of COVID-19, public health officials strongly recommended preventive measures such as disinfectants, alcohol-based hand sanitizers, and face masks. The effect of ethanol on virus structure and inactivation remains unclear, and its molecular mechanism needs to be elucidated. This study elucidates how ethanol solutions interact with the lipid bilayer of the COVID-19 virus utilizing molecular dynamics (MD) simulations. Its findings indicated that ethanol can deactivate the virus through two primary mechanisms. First, when ethanol penetrates the viral membrane, it disrupts the structural integrity of the lipid bilayer, leading to membrane disruption. This alteration in morphology is critical as it compromises the virus's ability to maintain its structure and function. For the simulation, a lipid bilayer containing the spike protein of SARS-CoV-2 was constructed. The interaction between the viral membrane and ethanol solution was then simulated using GROMACS 5.1.4 for molecular dynamics (MD) analysis. Also, visual molecular dynamics (VMD1.9.3) was used for visualization. The study calculated the Lennard-Jones (LJ) and electrostatic interactions between ethanol and the lipid bilayer, and it analyzed the conformational changes in the spike protein following ethanol adsorption. Additionally, the effects of ethanol penetration on the morphology of the lipid bilayer were evaluated.

Post-transfusion activation of coagulation pathways during severe COVID-19 correlates with COVID-19 convalescent plasma antibody profiles.

Early antibody therapy can prevent severe SARS-CoV-2 infection (COVID-19). However, the effectiveness of COVID-19 convalescent plasma (CCP) therapy in treating severe COVID-19 remains inconclusive. To test a hypothesis that some CCP units are associated with a coagulopathy hazard in severe disease that offsets its benefits, we tracked 304 CCP units administered to 414 hospitalized COVID-19 patients to assess their association with the onset of unfavorable post-transfusion D-dimer trends. CCP recipients with increasing or persistently elevated D-dimer trajectories after transfusion experienced higher mortality than those whose D-dimer levels were persistently low or decreasing after transfusion. Within the CCP donor-recipient network, recipients with increasing or persistently high D-dimer trajectories were skewed toward association with a minority of CCP units. In in vitro assays, CCP from "higher-risk" units had higher cross-reactivity with the spike protein of human seasonal betacoronavirus OC43. "Higher-risk" CCP units also mediated greater Fcγ receptor IIa signaling against cells expressing SARS-CoV-2 spike compared with "lower-risk" units. This study finds that post-transfusion activation of coagulation pathways during severe COVID-19 is associated with specific CCP antibody profiles and supports a potential mechanism of immune complex-activated coagulopathy.

Differential efficacy of first licensed western vaccines protecting without immunopathogenesis Wuhan-1-challenged hamsters from severe COVID-19.

Four COVID-19 vaccines were developed, tested, and authorized early in Europe and the US. Comirnaty and Spikevax are mRNA-based, whereas Jcovden and Vaxzevria utilize adenoviral vectors (AdV). We described a hamster model of COVID-19 utilizing Wuhan-1 strain SARS-CoV-2, in which vaccine-associated immunopathogenesis can be induced by Alum-adjuvanted Spike protein (Alum+S). Such animals were vaccinated with the authorized vaccines or Alum+S, challenged, and examined. All vaccinated hamsters produced antibodies targeting S. Neutralizing antibodies (nAb) were induced only by authorized vaccines. While nAbs were present after one vaccination with AdV-vaccines, mRNA vaccines needed a boost immunization. Upon challenge, all authorized vaccines protected from severe disease. Less tissue damage and no live virus (one exception) were detectable in the lungs. In contrast, Alum+S immunized hamsters developed VAERD. Our data reveal the absence of induction of VAERD by early commercial vaccines in hamsters, while animals´ immune responses and protection seem to match the clinical vaccine efficacy.

Correction: A promising mRNA vaccine derived from the JN.1 spike protein confers protective immunity against multiple emerged Omicron variants

Correction: A promising mRNA vaccine derived from the JN.1 spike protein confers protective immunity against multiple emerged Omicron variants

Using Nano-Luciferase Binary (NanoBiT) Technology to Assess the Interaction Between Viral Spike Protein and Angiotensin-Converting Enzyme II by Aptamers

Nano-luciferase binary technology (NanoBiT)-based pseudoviral sensors are innovative tools for monitoring viral infection dynamics. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells via its trimeric surface spike protein, which binds to the human angiotensin-converting enzyme II (hACE2) receptor. This interaction is crucial for viral entry and serves as a key target for therapeutic interventions against coronavirus disease 2019 (COVID-19). Aptamers, short single-stranded DNA (ssDNA) or RNA molecules, are highly specific, high-affinity biorecognition elements for detecting infective pathogens. Despite their potential, optimizing viral infection assays using traditional protein–protein interaction (PPI) methods often face challenges in optimizing viral infection assays. In this study, we selected and evaluated aptamers for their ability to interact with viral proteins, enabling the dynamic visualization of infection progression. The NanoBiT-based pseudoviral sensor demonstrated a rapid increase in luminescence within 3 h, offering a real-time measure of viral infection. A comparison of detection technologies, including green fluorescent protein (GFP), luciferase, and NanoBiT technologies for detecting PPI between the pseudoviral spike protein and hACE2, highlighted NanoBiT’s superior sensitivity and performance, particularly in aptamer selection. This bioluminescent system provides a robust, sensitive, and early-stage quantitative approach to studying viral infection dynamics.

Immunological assessment of NSFu1: A novel fusion molecule constructed from structural proteins of SARS-CoV-2 for improving COVID-19 antibody detection.

The SARS-CoV-2 outbreak has claimed millions of lives and caused significant clinical challenges. The availability of a rapid, cost-effective, and sensitive test to detect antibodies at different stages of COVID-19 is crucial for effective clinical management, epidemiological studies, and public health surveillance. Four novel peptides (SF1, SF2, SF4, SF6) and two multi-epitope fusion proteins (SFu1 and NSFu1) from less variable regions of the spike and nucleocapsid proteins were developed. After detailed in silico structural validation, all the proteins were expressed in E. coli (BL21), purified by Ni2+ affinity chromatography, and CD spectroscopy was also executed for secondary structural analysis. The serological potential was assessed by screening 462 plasma samples from symptomatic, asymptomatic, recovered, follow-up COVID-19 cases, and 212 healthy controls. The recombinant antigens SF1, SF2, SF4, SF6, NC, SFu1, and NSFu1 showed ELISA sensitivities of 32.9%, 41.5%, 37.3%, 28.8%, 30.7%, 65.8%, and 82.0%, respectively with specificities ranging from 97 to 99% for symptomatic and asymptomatic COVID-19 cases. The sensitivities for the fusion proteins were nearly equivalent to the combined sensitivities of their constituent antigens. In conclusion, the NSFu1 fusion protein showing 82% sensitivity and 99% specificity could be a potential antigen for developing new molecules to achieve higher sensitivity for COVID-19 antibody detection.

Bovine coronavirus presence in domestic bovine and antelopes sub-Saharan Africa: evidence from Namibia

BackgroundBovine coronavirus (BoCV) causes significant economic losses to cattle farming due to mortality in calves, reduced growth performances and milk production in feedlots and dairy cattle. Worldwide distribution of BoCV has been demonstrated, although knowledge of its epidemiology in Africa, especially in the sub-Saharan region, is limited.ResultsIn the present study, a total of 208 swab samples of wild ruminants and 435 bovines from different regions of Namibia were obtained and tested by a BoCV-specific qRT-PCR. Twenty-six bovine samples tested positive [26/435 (5.98%; 95CI: 3.94-8.64%)] while, among the wild ruminants, only Greater Kudu (Tragelaphus strepsiceros) were shown to be positive [13/52 (25.00%; 95CI: 14.03-38.95%)] of which 8 showed clinical signs. Analysis of partial nucleoprotein and spike protein gene sequences and comparison with international reference sequences demonstrated the existence of a unique Namibian clade, resulting from a single introduction event around 2010 followed by local evolution. Although the introduction source remains unknown, contact between bovine and wild animals appears likely.ConclusionsThe present study represents the first report of BoCV circulation in southern Africa, which showed a relatively high frequency and the ability of persisting and evolving locally in the absence of further foreign introductions. The implications for disease spread among domestic bovines and the potential impact on wildlife should encourage broader investigations on BoCV involving other African countries. Moreover, the Greater Kudu’s susceptibility to BoCV infection was also proven, further highlighting the host plasticity of this virus.

Effects of different sources of lactoferrin on cytokine response to SARS-COV-2, respiratory syncytial virus, and rotavirus infection in vitro.

Lactoferrin (Lf) is a multifunctional iron-binding glycoprotein, involved in a wide range of bioactivities, including immunomodulatory and antiviral activities. Lf in human milk and bovine Lf added to infant formula may provide some protection against viral infections. However, functions of Lfs from different sources may differ due to varying manufacturing processes and posttranslational modifications. Here, effects of Lfs (11 commercial bovine milk Lfs, 2 recombinant Lfs, and native human/bovine milk Lf) on cytokine responses to virus infection were examined by infecting human intestinal epithelial cells (Caco-2 cells) with rotavirus (naked) or normal human bronchial epithelial cells (BEAS-2B cells) with respiratory syncytial virus (RSV, enveloped) or SARS-CoV-2 spike protein 1. Effects of Lf on viral infection were evaluated by qRT-PCR analysis of transcripts of cytokines/chemokines (TNF-α, IL-1 β, IL-6, IL-8, IL-10, IFN-β, and CXCL10). Our results show that viral infection changes transcription of these cytokines and that Lfs significantly and variously influence immune responses to rotavirus, RSV, and SARS-CoV-2 in vitro. Thus, Lf may provide protection against virus infection by down-regulating pro‑inflammatory cytokine/chemokine responses. Recombinant bovine and human Lf show similar effects as bovine milk Lfs suggesting that different posttranslational modifications do not affect the antiviral activity on cytokine response.

A competitive ELISA based on nanobodies for the detection of serum neutralizing antibodies against porcine epidemic diarrhea virus

Porcine epidemic diarrhea (PED), caused by porcine epidemic diarrhea virus (PEDV), can induce 80–100% mortality in newborn piglets; therefore, specific and rapid detection methods are important for the prevention of this viral infection. In particular, methods for detecting neutralizing antibodies (nAbs) can be used to evaluate the immunization effect of PEDV vaccines. The spike protein of PEDV (PEDV-S) has been universally used as an antigen to develop immunoassays to detect nAbs. Nanobodies (Nbs) offer advantages such as ease of genetic engineering and low production costs, making them promising for diagnostic applications. In this study, PEDV-S was expressed via the baculovirus system and was used as an antigen to immunize Bactrian camels. A total of 10 Nbs against PEDV-S were first screened and expressed as fusion proteins with horseradish peroxidase (HRP) in HEK293T cells. A Nb-HRP fusion protein named PEDV-S-Nb13-HRP was subsequently selected and used as a probe for developing a competitive enzyme-linked immunosorbent assay (cELISA) to detect anti-PEDV nAbs. Optimization assays identified 80 ng/well of PEDV-S as the optimal coating antigen concentration. The optimal dilution of PEDV-S-Nb13-HRP was 1:200, and the optimal serum dilution was 1:10. The cutoff value of cELISA was determined as 28.1%, demonstrating high specificity, repeatability, stability, and good agreement rates with two commercial ELISA kits (93.6%) and a serum neutralization test (96.34%). Additionally, the results of the detection of IgA antibodies in oral and milk samples from sows were in good agreement with those of the IDEXX PEDV IgA kit. These results demonstrate that the cELISA is a reliable and cost-effective method for detecting anti-PEDV nAbs.

Poly(3,4-ethylenedioxythiophene) Nanorod Arrays-Based Organic Electrochemical Transistor for SARS-CoV-2 Spike Protein Detection in Artificial Saliva.

The outbreak and continued spread of coronavirus disease 2019 (COVID-19) have significantly threatened public health. Antibody testing is essential for infection diagnosis, seroepidemiological analysis, and vaccine evaluation. However, achieving convenient, fast, and accurate detection remains challenging in this prolonged battle. This study reports a highly sensitive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein detection platform based on organic electrochemical transistors (OECTs) for biosensing applications. We developed a nanostructured poly(3,4-ethylenedioxythiophene) (PEDOT) conductive polymer with the carboxylic acid functional group (PEDOTAc) for modifying specific antibodies on an OECT channel for the detection of the COVID-19 spike protein. The OECT device features a channel composed of a PEDOT:polystyrenesulfonate (PEDOT:PSS) bottom layer, with the upper layer decorated with PEDOTAc nanorod arrays via the oxidative polymerization and a trans-printing method. Our novel PEDOTAc nanorod array-based OECT device exhibits promising potential for future healthcare and point-of-care sensing due to its rapid response, high sensitivity, and high accuracy. Through optimization, we achieved specific detection of the SARS-CoV-2 spike protein within minutes, with a detectable region from 10 fM to 100 nM. These biosensors hold significant promise for use in the diagnosis and prognosis of COVID-19.

Potent bivalent nanobody constructs that protect against the SARS-CoV-2 XBB variant

Most antibody-based therapeutics approved for SARS-CoV-2 treatment have shown greatly reduced neutralization activity against emerging Omicron variants. To target recent Omicron variants, we developed XBB-specific antibody-like therapeutics by screening a yeast surface-displayed single-domain antibody library against the receptor binding domain of the XBB spike protein. Three lead nanobodies, XNb 4.13, XNb 4.14, and XNb 4.15, were selected based on their binding affinity to the XBB spike protein and were fused to a mouse Fc domain. While all three constructs showed sub-nanomolar binding affinities to the XBB S protein, XNb 4.13-Fc and XNb 4.14-Fc also neutralized XBB in vitro. Intraperitoneal injection of XNb 4.13-Fc and XNb 4.14-Fc and intranasal delivery of XNb 4.13-Fc protected transgenic mice from a challenge with XBB virus with a significant reduction in viral lung titers post-infection. These antibody-like constructs identified using yeast surface display have potential as therapeutics that can protect against SARS-CoV-2 XBB variants.