Spike Protein Variant Research Articles

Effects of Recombinant SARS-CoV-2 Spike Protein Variants on Platelet Morphology and Activation.

Platelets are central elements of hemostasis and also play a pivotal role in the pathogenesis of thrombosis in coronavirus disease 2019. This study was planned to investigate the effects of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recombinant spike protein variants on platelet morphology and activation. Citrated whole blood collected from ostensibly healthy subjects was challenged with saline (control sample) and with 2 and 20 ng/mL final concentration of SARS-CoV-2 recombinant spike protein of Ancestral, Alpha, Delta, and Omicron variants. Platelet count was found to be decreased with all SARS-CoV-2 recombinant spike protein variants and concentrations tested, achieving the lowest values with 20 ng/mL Delta recombinant spike protein. The mean platelet volume increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, but especially using Delta and Alpha recombinant spike proteins. The values of both platelet function analyzer-200 collagen-adenosine diphosphate and collagen-epinephrine increased in all samples irrespective of SARS-CoV-2 recombinant spike protein variants and concentrations tested, and thus reflecting platelet exhaustion, and displaying again higher increases with Delta and Alpha recombinant spike proteins. Most samples where SARS-CoV-2 recombinant spike proteins were added were flagged as containing platelet clumps. Morphological analysis revealed the presence of a considerable number of activated platelets, platelet clumps, platelet-monocyte, and platelet-neutrophils aggregates, especially in samples spiked with Alpha and Delta recombinant spike proteins at 20 ng/mL. These results provide support to the evidence that SARS-CoV-2 is capable of activating platelets through its spike protein, though such effect varies depending on different spike protein variants.

SARS CoV-2 spike protein variants exploit DC-SIGN/DC-SIGNR receptor for evolution and severity: an in-silico insight.

The online version contains supplementary material available at 10.1007/s13337-023-00820-3.

Analysis of SARS-CoV-2 Spike Protein Variants with Recombinant Reporter Viruses Created from a Bacmid System.

SARS-CoV-2, the causative agent of COVID-19, has spread around the world with more than 700 million cases and 6.8 million deaths. Various variants of concern (VoC) have emerged due to mutations and recombination and concurrent selection for increased viral fitness and immune evasion. The viral protein that primarily determines the pathogenicity, infectivity, and transmissibility is the Spike protein. To analyze the specific impact of variant Spike proteins on infection dynamics, we constructed SARS-CoV-2 with a uniform B.1 backbone but with alternative Spike proteins. In addition, ORF6 was replaced by EYFP as a biological safety measure, and for use of this well-established reporter. We show that namely the delta variant Spike proteins cause a distinct phenotype from the wild type (B.1, D614G) and other variants of concern. Furthermore, we demonstrate that the omicron BA.1 Spike results in lower viral loads and a less efficient spread in vitro. Finally, we utilized viruses with the two different reporters EYFP and mCherry to establish a competitive growth assay, demonstrating that most but not all Spike variant viruses were able to outcompete wild type SARS-CoV-2 B.1.

High-Affinity Neutralizing DNA Aptamers against SARS-CoV-2 Spike Protein Variants

The continuous emergence of new variants of concern for SARS-CoV-2 has created a challenge for existing therapies. To address this, we developed a series of single-stranded DNA aptamers that not only bind specifically to the trimer S protein of SARS-CoV-2 but also block the interaction between the trimer S protein and ACE2 receptors. The systematic evolution of ligands by exponential enrichment (SELEX) was performed to select the aptamers for SARS-CoV-2 trimer S protein. ELISA-based assay and flow cytometry were performed to test the apatmers’ binding and inhibition of trimer S protein in vitro. Binding affinity was measured using surface plasmon resonance. Significance was determined in Prism 9.0 using the one-way ANOVA test (Dunnett’s multiple comparisons test) or two-way ANOVA test (Tukey’s multiple comparisons test) for comparisons. The p values < 0.05 were considered statistically significant. After 12 rounds of SELEX, eight highly enriched aptamers were able to bind to the trimer S protein of the SARS-CoV-2 Wuhan original strain as well as the trimer S proteins of the Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants, with affinities in the nM range, while also inhibiting their interaction with ACE2 receptors in Vero E6 cells. Modifications to our best aptamer were made by adding forward and reverse primer sequences and truncation. The modified aptamers AYA2012004_L and AYA2012004_L-M1 showed up to 70% inhibition of the binding of virus-like particles (VLPs) expressing S protein to the ACE2 receptor expressed in HEK293T cells. Our findings imply that the selected aptamers can prevent SARS-CoV-2 from entering host cells and hence suppress the viral infection. In addition, the findings suggest that the selected aptamers might be an innovative therapy for the treatment of COVID-19.

Serologic responses to COVID-19 vaccination in children with history of multisystem inflammatory syndrome (MIS-C).

Understanding the serological responses to COVID-19 vaccination in children with history of MIS-C could inform vaccination recommendations. We prospectively enrolled seven children hospitalized with MIS-C and measured SARS-CoV-2 binding IgG antibodies to spike protein variants longitudinally pre- and post-Pfizer-BioNTech BNT162b2 primary series COVID-19 vaccination. We found that SARS-CoV-2 variant cross-reactive IgG antibodies variably waned following acute MIS-C, but were significantly boosted with vaccination and maintained for up to 3months. We then compared post-vaccination binding, pseudovirus neutralizing, and functional antibody-dependent cell-mediated cytotoxicity (ADCC) titers to the reference strain (Wuhan-hu-1) and Omicron variant (B.1.1.529) among previously healthy children (n=16) and children with history of MIS-C (n=7) or COVID-19 (n=8). Despite the breadth of binding antibodies elicited by vaccination in all three groups, pseudovirus neutralizing and ADCC titers were significantly reduced to the Omicron variant.

Computational design of nanomolar-binding antibodies specific to multiple SARS-CoV-2 variants by engineering a specificity switch of antibody 80R using RosettaAntibodyDesign (RAbD) results in potential generalizable therapeutic antibodies for novel SARS-CoV-2 virus

The human infectious disease COVID-19 caused by the SARS-CoV-2 virus has become a major threat to global public health. Developing a vaccine is the preferred prophylactic response to epidemics and pandemics. However, for individuals who have contracted the disease, the rapid design of antibodies that can target the SARS-CoV-2 virus fulfils a critical need. Further, discovering antibodies that bind multiple variants of SARS-CoV-2 can aid in the development of rapid antigen tests (RATs) which are critical for the identification and isolation of individuals currently carrying COVID-19. Here we provide a proof-of-concept study for the computational design of high-affinity antibodies that bind to multiple variants of the SARS-CoV-2 spike protein using RosettaAntibodyDesign (RAbD). Well characterized antibodies that bind with high affinity to the SARS-CoV-1 (but not SARS-CoV-2) spike protein were used as templates and re-designed to bind the SARS-CoV-2 spike protein with high affinity, resulting in a specificity switch. A panel of designed antibodies were experimentally validated. One design bound to a broad range of variants of concern including the Omicron, Delta, Wuhan, and South African spike protein variants.

Abiotic Synthetic Antibody Inhibitor with Broad-Spectrum Neutralization and Antiviral Efficacy against Escaping SARS-CoV-2 Variants.

The rapid emergence and spread of vaccine/antibody-escaping variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed serious challenges to our efforts in combating corona virus disease 2019 (COVID-19) pandemic. A potent and broad-spectrum neutralizing reagent against these escaping mutants is extremely important for the development of strategies for the prevention and treatment of SARS-CoV-2 infection. We herein report an abiotic synthetic antibody inhibitor as a potential anti-SARS-CoV-2 therapeutic agent. The inhibitor, Aphe-NP14, was selected from a synthetic hydrogel polymer nanoparticle library created by incorporating monomers with functionalities complementary to key residues of the SARS-CoV-2 spike glycoprotein receptor binding domain (RBD) involved in human angiotensin-converting enzyme 2 (ACE2) binding. It has high capacity, fast adsorption kinetics, strong affinity, and broad specificity in biologically relevant conditions to both the wild type and the current variants of concern, including Beta, Delta, and Omicron spike RBD. The Aphe-NP14 uptake of spike RBD results in strong blockage of spike RBD-ACE2 interaction and thus potent neutralization efficacy against these escaping spike protein variant pseudotyped viruses. It also inhibits live SARS-CoV-2 virus recognition, entry, replication, and infection in vitro and in vivo. The Aphe-NP14 intranasal administration is found to be safe due to its low in vitro and in vivo toxicity. These results establish a potential application of abiotic synthetic antibody inhibitors in the prevention and treatment of the infection of emerging or possibly future SARS-CoV-2 variants.

Highly Selective Aptamer-Molecularly Imprinted Polymer Hybrids for Recognition of SARS-CoV-2 Spike Protein Variants.

Virus recognition has been driven to the forefront of molecular recognition research due to the COVID-19 pandemic. Development of highly sensitive recognition elements, both natural and synthetic is critical to facing such a global issue. However, as viruses mutate, it is possible for their recognition to wane through changes in the target substrate, which can lead to detection avoidance and increased false negatives. Likewise, the ability to detect specific variants is of great interest for clinical analysis of all viruses. Here, a hybrid aptamer-molecularly imprinted polymer (aptaMIP), that maintains selective recognition for the spike protein template across various mutations, while improving performance over individual aptamer or MIP components (which themselves demonstrate excellent performance). The aptaMIP exhibits an equilibrium dissociation constant of 1.61 nM toward its template which matches or exceeds published examples of imprinting of the spike protein. The work here demonstrates that "fixing" the aptamer within a polymeric scaffold increases its capability to selectivity recognize its original target and points toward a methodology that will allow variant selective molecular recognition with exceptional affinity.

Conformational Behavior of SARS-Cov-2 Spike Protein Variants: Evolutionary Jumps in Sequence Reverberate in Structural Dynamic Differences.

SARS-CoV-2 has evolved rapidly in the first 3 years of pandemic diffusion. The initial evolution of the virus appeared to proceed through big jumps in sequence changes rather than through the stepwise accumulation of point mutations on already established variants. Here, we examine whether this nonlinear mutational process reverberates in variations of the conformational dynamics of the SARS-CoV-2 Spike protein (S-protein), the first point of contact between the virus and the human host. We run extensive microsecond-scale molecular dynamics simulations of seven distinct variants of the protein in their fully glycosylated state and set out to elucidate possible links between the mutational spectrum of the S-protein and the structural dynamics of the respective variant, at global and local levels. The results reveal that mutation-dependent structural and dynamic modulations mostly consist of increased coordinated motions in variants that acquire stability and in an increased internal flexibility in variants that are less stable. Importantly, a limited number of functionally important substructures (the receptor binding domain, in particular) share the same time of movements in all variants, indicating efficient preorganization for functional regions dedicated to host interactions. Our results support a model in which the internal dynamics of the S-proteins from different strains varies in a way that reflects the observed random and non-stepwise jumps in sequence evolution, while conserving the functionally oriented traits of conformational dynamics necessary to support productive interactions with host receptors.

MERS virus spike protein HTL-epitopes selection and multi-epitope vaccine design using computational biology

MERS-CoV, a zoonotic virus, poses a serious threat to public health globally. Thus, it is imperative to develop an effective vaccination strategy for protection against MERS-CoV. Immunoinformatics and computational biology tools provide a faster and more cost-effective strategy to design potential vaccine candidates. In this work, the spike proteins from different strains of MERS-CoV were selected to predict HTL-epitopes that show affinity for T-helper MHC-class II HTL allelic determinant (HLA-DRB1:0101). The antigenicity and conservation of these epitopes among the selected spike protein variants in different MERS-CoV strains were analyzed. The analysis identified five epitopes with high antigenicity: QSIFYRLNGVGITQQ, DTIKYYSIIPHSIRS, PEPITSLNTKYVAPQ, INGRLTTLNAFVAQQ and GDMYVYSAGHATGTT. Then, a multi-epitope vaccine candidate was designed using linkers and adjuvant molecules. Finally, the vaccine construct was subjected to molecular docking with TLR5 (Toll-like receptor-5). The proposed vaccine construct had strong binding energy of −32.3 kcal/mol when interacting with TLR5.Molecular dynamics simulation analysis showed that the complex of the vaccine construct and TLR5 is stable. Analysis using in silico immune simulation also showed that the prospective multi-epitope vaccine design had the potential to elicit a response within 70 days, with the immune system producing cytokines and immunoglobulins. Finally, codon adaptation and in silico cloning analysis showed that the candidate vaccine could be expressed in the Escherichia coli K12 strain. Here we also designed support vaccine construct MEV-2 by using B-cell and CD8+ CTL epitopes to generate the complete immunogenic effect. This study opens new avenues for the extension of research on MERS vaccine development. Communicated by Ramaswamy H. Sarma

South African (501Y.V2) and the United Kingdom (B.1.1.7) SARS-CoV-2 Spike (S) Protein Variants Demonstrate a Higher Binding Affinity to ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) affects the lower respiratory tract by binding to angiotensin-converting enzyme 2 (ACE2) via its S-protein. Recent emerging SARS-CoV-2 variants from the United Kingdom (B.1.1.7) and South Africa (501Y.V2) are spreading worldwide at an alarming rate. The new variants have manifested amino acid substitution K417N, E484K, and N501Y on the RBD domain that binds to ACE2. These mutations may influence the binding of the S-protein to ACE2 and affect viral entry into the host cell. In this study, we modelled the amino acid substitutions on the S-protein and utilised the HADDOCK server to assess the S-protein RBD domain binding with ACE2. Additionally, we calculated the binding affinity of ACE2 to S-protein WT, B.1.1.7 and 501Y.V2 variants using Molecular Mechanics-Generalized Born Surface Area (MM/GBSA). We demonstrated that the S-protein of both variants possesses a higher binding affinity to ACE2 than WT, with the South African 501Y.V2 being more infective than the B.1.1.7 that originated in the United Kingdom. The South African 501Y.V2 variant presents three amino acid substitutions that changed the H-bonding network, resulting in a higher affinity to ACE2, indicating that the 501Y.V2 strain is more infective than the B.1.1.7 strain.

Trajectories of Seroprevalence and Neutralizing Activity of Antibodies against SARS-CoV-2 in Southern Switzerland between July 2020 and July 2021: An Ongoing, Prospective Population-Based Cohort Study.

The COVID-19 pandemic continues, and evidence on infection- and vaccine-induced immunity is key. We assessed COVID-19 immunity and the neutralizing antibody response to virus variants across age groups in the Swiss population. We conducted a cohort study in representative community-dwelling residents aged five years or older in southern Switzerland (total population 353,343), and we collected blood samples in July 2020 (in adults only, N = 646), November-December 2020 (N = 1457), and June-July 2021 (N = 885). We used a previously validated Luminex assay to measure antibodies targeting the spike (S) and the nucleocapsid (N) proteins of the virus and a high-throughput cell-free neutralization assay optimized for multiple spike protein variants. We calculated seroprevalence with a Bayesian logistic regression model accounting for the population's sociodemographic structure and the test performance, and we compared the neutralizing activity between vaccinated and convalescent participants across virus variants. The overall seroprevalence was 7.8% (95% CI: 5.4-10.4) by July 2020 and 20.2% (16.4-24.4) by December 2020. By July 2021, the overall seroprevalence increased substantially to 72.5% (69.1-76.4), with the highest estimates of 95.6% (92.8-97.8) among older adults, who developed up to 10.3 more antibodies via vaccination than after infection compared to 3.7 times more in adults. The neutralizing activity was significantly higher for vaccine-induced than infection-induced antibodies for all virus variants (all p values < 0.037). Vaccination chiefly contributed to the reduction in immunonaive individuals, particularly those in older age groups. Our findings on the greater neutralizing activity of vaccine-induced antibodies than infection-induced antibodies are greatly informative for future vaccination campaigns.

Distinct Conformations of SARS-CoV-2 Omicron Spike Protein and Its Interaction with ACE2 and Antibody.

Since November 2021, Omicron has been the dominant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant that causes the coronavirus disease 2019 (COVID-19) and has continuously impacted human health. Omicron sublineages are still increasing and cause increased transmission and infection rates. The additional 15 mutations on the receptor binding domain (RBD) of Omicron spike proteins change the protein conformation, enabling the Omicron variant to evade neutralizing antibodies. For this reason, many efforts have been made to design new antigenic variants to induce effective antibodies in SARS-CoV-2 vaccine development. However, understanding the different states of Omicron spike proteins with and without external molecules has not yet been addressed. In this review, we analyze the structures of the spike protein in the presence and absence of angiotensin-converting enzyme 2 (ACE2) and antibodies. Compared to previously determined structures for the wildtype spike protein and other variants such as alpha, beta, delta, and gamma, the Omicron spike protein adopts a partially open form. The open-form spike protein with one RBD up is dominant, followed by the open-form spike protein with two RBD up, and the closed-form spike protein with the RBD down. It is suggested that the competition between antibodies and ACE2 induces interactions between adjacent RBDs of the spike protein, which lead to a partially open form of the Omicron spike protein. The comprehensive structural information of Omicron spike proteins could be helpful for the efficient design of vaccines against the Omicron variant.

Towards Quantum-Chemical Level Calculations of SARS-CoV-2 Spike Protein Variants of Concern by First Principles Density Functional Theory.

The spike protein (S-protein) is a crucial part of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with its many domains responsible for binding, fusion, and host cell entry. In this review we use the density functional theory (DFT) calculations to analyze the atomic-scale interactions and investigate the consequences of mutations in S-protein domains. We specifically describe the key amino acids and functions of each domain, which are essential for structural stability as well as recognition and fusion processes with the host cell; in addition, we speculate on how mutations affect these properties. Such unprecedented large-scale ab initio calculations, with up to 5000 atoms in the system, are based on the novel concept of amino acid-amino acid-bond pair unit (AABPU) that allows for an alternative description of proteins, providing valuable information on partial charge, interatomic bonding and hydrogen bond (HB) formation. In general, our results show that the S-protein mutations for different variants foster an increased positive partial charge, alter the interatomic interactions, and disrupt the HB networks. We conclude by outlining a roadmap for future computational research of biomolecular virus-related systems.

Longitudinal IgA and IgG Response, and ACE2 Binding Blockade, to Full-Length SARS-CoV-2 Spike Protein Variants in a Population of Black PLWH Vaccinated with ChAdOx1 nCoV-19.

Vaccines against SARS-CoV-2 have been pivotal in overcoming the COVID-19 pandemic yet understanding the subsequent outcomes and immunological effects remain crucial, especially for at-risk groups e.g., people living with human immunodeficiency virus (HIV) (PLWH). In this study we report the longitudinal IgA and IgG antibody titers, as well as antibody-mediated angiotensin converting enzyme 2 (ACE2) binding blockade, against the SARS-CoV-2 spike (S) proteins after 1 and 2 doses of the ChAdOx1 nCoV-19 vaccine in a population of Black PLWH. Here, we report that PLWH (N = 103) did not produce an anti-S IgA response after infection or vaccination, however, anti-S IgG was detected in response to vaccination and infection, with the highest level detected for infected vaccinated participants. The anti-IgG and ACE2 blockade assays revealed that both vaccination and infection resulted in IgG production, however, only vaccination resulted in a moderate increase in ACE2 binding blockade to the ancestral S protein. Vaccination with a previous infection results in the greatest anti-S IgG and ACE2 blockade for the ancestral S protein. In conclusion, PLWH produce an anti-S IgG response to the ChAdOx1 nCoV-19 vaccine and/or infection, and ChAdOx1 nCoV-19 vaccination with a previous infection produced more neutralizing antibodies than vaccination alone.

Development of a Series of Neutralizing Nanobodies against SARS-CoV-2 Spike Protein

Countering the spread of new respiratory infections and reducing the damage they cause to society requires efficient strategies for rapidly developing of targeted therapeutics, such as monoclonal antibodies. Nanobodies, defined as variable fragments of heavy-chain camelid antibodies, have a set of characteristics that make them particularly convenient for this purpose. The speed at which the SARS-CoV-2 pandemic spread confirmed that the key factor in the development of therapeutics is obtaining highly effective blocking agents as soon as possible, as well as the diversity of epitopes to which these agents bind. We have optimized the selection process of blocking nanobodies from the genetic material of camelids and obtained a panel of nanobody structures with affinity to Spike protein in the lower nanomolar and picomolar ranges and with high binding specificity. The subset of nanobodies that demonstrate the ability to block the interaction between the Spike protein and the cellular ACE2 receptor was selected in experiments in vitro and in vivo. It has been established that the epitopes bound by the nanobodies are located in the RBD domain of the Spike protein and have little overlap. The diversity of binding regions may allow a mixture of nanobodies to retain potential therapeutic efficacy towards new Spike protein variants. Furthermore, the structural features of nanobodies, particularly their compact size and high stability, indicate the possibility of their utilization in the form of aerosols.

Quantitative profiling of N-glycosylation of SARS-CoV-2 spike protein variants.

With the global spread of the corona virus disease-2019 pandemic, new spike variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continuously emerge due to increased possibility of virus adaptive amino acid mutations. However, the N-glycosylation profiles of different spike variants are yet to be explored extensively, although the spike protein is heavily glycosylated and surface glycans are well-established to play key roles in viral infection and immune response. Here, we investigated quantitatively the N-glycosylation profiles of seven major emerging spike variants including Original, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.671.1), Delta (B.1.671.2), and Omicron (B.1.1.529). The aim was to understand the changing pattern of N-glycan profiles in SARS-CoV-2 evolution in addition to the widely studied amino acid mutations. Different spike variants exhibit substantial variations in the relative abundance of different glycan peaks and subclasses, although no specific glycan species are exclusively present in or absent from any specific variant. Cluster analysis shows that the N-glycosylation profiles may hold the potential for SARS-CoV-2 spike variants classification. Alpha and Beta variants exhibit the closest similarity to the Original, and the Delta variant displays substantial similarity to Gamma and Kappa variants, while the Omicron variant is significantly different from its counterparts. We demonstrated that there is a quantifiable difference in N-glycosylation profiles among different spike variants. The current study and observations herein provide a valuable framework for quantitative N-glycosylation profiling of new emerging viral variants and give us a more comprehensive picture of COVID-19 evolution.

Integration of smFRET and molecular dynamics to characterize conformational dynamics of the spike protein of wild-type SARS-CoV-2 and its variants.

Since the rise of the COVID-19 pandemic, researchers have pushed for development of efficient therapeutics and vaccines to assist in preventing the severity of its viral effects. A major target has been the trimeric SARS-CoV-2 spike protein. There has been various experimental and computational efforts to characterize the structural dynamics of this protein at different stages of its mechanisms. Among these methods are single molecule fluorescence resonance energy transfer (smFRET) experiments as well as molecular dynamics (MD) simulations. The purpose of our current work here is to combine the existing data from smFRET experiments and MD simulations to provide a more complete picture of prefusion SARS-CoV-2 spike protein conformational dynamics. We have particularly examined the conformational dynamics of several variants of this protein in a comparative manner. Specifically, we have examined multiple computational methods based on “implicit screening” approach within different approximations to predict dye-dye distance and FRET efficiency distributions to provide reliable interpretations for both smFRET experimental data and MD simulation data of various spike protein variants including the wild-type, the alpha, beta, gamma, delta, and omega variants as well as the engineered spike protein used in the Moderna vaccine. This work sheds light on the structural heterogeneity of the spike protein in different variants of SARS-CoV-2.

Determining the Optimal SARS-CoV-2 mRNA Vaccine Dosing Interval for Maximum Immunogenicity.

Emerging evidence indicates that longer SARS-CoV-2 vaccine dosing intervals results in an enhanced immune response. However, the optimal vaccine dosing interval for achieving maximum immunogenicity is unclear. This study included samples from adult paramedics in Canada who received two doses of either BNT162b2 or mRNA-1273 vaccines and provided blood samples six months (170 to 190 days) after the first vaccine dose. The main exposure variable was vaccine dosing interval (days), categorized as "short" (first quartile), "moderate" (second quartile), "long" (third quartile), and "longest" interval (fourth quartile). The primary outcome was total spike antibody concentrations, measured using the Elecsys SARS-CoV-2 total antibody assay. Secondary outcomes included spike and receptor-binding domain (RBD) immunoglobulin G (IgG) antibody concentrations, and inhibition of angiotensin-converting enzyme 2 (ACE-2) binding to wild-type spike protein and several different Delta variant spike proteins. We fit a multiple log-linear regression model to investigate the association between vaccine dosing intervals and the antibody concentrations. A total of 564 adult paramedics (mean age 40 years, SD=10) were included. Compared to "short interval" (≤30 days), vaccine dosing intervals of the long (39-73 days) group (β= 0.31, 95% Confidence interval (CI): 0.10-0.52) and the longest (≥74 days) group (β = 0.82. 95% CI: 0.36-1.28) were associated with increased spike total antibody concentration. Compared to the short interval, the longest interval quartile was associated with higher spike IgG antibodies, while the long and longest intervals were associated with higher RBD IgG antibody concentrations. Similarly, the longest dosing intervals increased inhibition of ACE-2 binding to viral spike protein. Increased mRNA vaccine dosing intervals longer than 38 days result in higher levels of anti-spike antibodies and ACE-2 inhibition when assessed six months after the first COVID-19 vaccine.

Glycopeptide characterization of Sf9-derived SARS-CoV-2 spike protein recombinant vaccine candidates expedited by the use of glycopeptide libraries.

We report the N-glycosylation pattern of Sf9 insect cell-derived recombinant spike proteins being developed as candidate vaccine antigens for SARS-CoV-2 (COVID-19) (Sanofi). The method has been optimised to produce peptides with single, isolated glycosylation sites using multiple protease digests. The development and use of glycopeptide libraries from previous developmental phases allowed for faster analysis than processing datasets from individual batches from first principles. Purified spike proteins were reduced, alkylated, and digested with proteolytic enzymes. Three different protease digests were utilised to generate peptides with isolated glycosylation sites. The glycopeptides were then analysed using a Waters Q-TOF while using a data-dependent acquisition mass spectrometry experiment. Glycopeptide mapping data processing and glycan classification were performed using Genedata Expressionist via a specialised workflow that used libraries of previously detected glycopeptides to greatly reduce processing time. Two different spike proteins from six manufacturers were analysed. There was a strong similarity at each site across batches and manufacturers. The majority of the glycans present were of the truncated class, although at sites N61, N234, and N717/714 high mannose structures were dominant and at N1173/1170 aglycosylation was dominant for both variant proteins. A comparison was performed on a commercially available spike protein and our results were found to be similar to those of earlier reports. Our data clearly show that the overall glycosylation pattern of both spike protein variants was highly similar from batch to batch, and between materials produced at different manufacturing facilities. The use of our glycopeptide libraries greatly expedited the generation of site-specific glycan occupancy data for a large glycoprotein. We compared our method with previously obtained data from a commercially available insect cell-derived spike protein and the results were comparable to published findings.