In Silico Comparative Analysis of the Functional, Structural, and Evolutionary Properties of SARS-CoV-2 Variant Spike Proteins.

Background: A recent global outbreak of COVID-19 caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) created a pandemic and emerged as a potential threat to humanity. The analysis of virus genetic composition has revealed that the spike protein, one of the major structural proteins, facilitates the entry of the virus to host cells. Objective: The spike protein has become the main target for prophylactics and therapeutics studies. Here, we compared the spike proteins of SARS-CoV-2 variants using bioinformatics tools. Methods: The spike protein sequences of wild-type SARS-CoV-2 and its 6 variants-D614G, alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), gamma (P.1), and omicron (B.1.1.529)-were retrieved from the NCBI database. The ClustalX program was used to sequence multiple alignment and perform mutational analysis. Several online bioinformatics tools were used to predict the physiological, immunological, and structural features of the spike proteins of SARS-CoV-2 variants. A phylogenetic tree was constructed using CLC software. Statistical analysis of the data was done using jamovi 2 software. Results: Multiple sequence analysis revealed that the P681R mutation in the delta variant, which changed an amino acid from histidine (H) to arginine (R), made the protein more alkaline due to arginine's high pKa value (12.5) compared to histidine's (6.0). Physicochemical properties revealed the relatively higher isoelectric point (7.34) and aliphatic index (84.65) of the delta variant compared to other variants. Statistical analysis of the isoelectric point, antigenicity, and immunogenicity of all the variants revealed significant correlation, with P values ranging from <.007 to .04. The generation of a 2D gel map showed the separation of the delta spike protein from a grouping of the other variants. The phylogenetic tree of the spike proteins showed that the delta variant was close to and a mix of the Rousettus bat coronavirus and MERS-CoV. Conclusions: The comparative analysis of SARS-CoV-2 variants revealed that the delta variant is more aliphatic in nature, which provides more stability to it and subsequently influences virus behavior.

SARS-CoV-2 variants and spike mutations involved in second wave of COVID-19 pandemic in India.

Against the backdrop of the second wave of COVID-19 pandemic in India that started in March 2021, we have monitored the spike (S) protein mutations in all the reported (GISAID portal) whole-genome sequences of SARS-CoV-2 circulating in India from 1 January 2021 to 31 August 2021. In the 43,102 SARS-CoV-2 genomic sequences analysed, we have identified 24,260 amino acid mutations in the S protein, based on which 265 Pango lineages could be categorized. The dominant lineage in most of the 28 states of India and its 8 union territories was B.1.617.2 (the delta variant). However, the states Madhya Pradesh, Jammu & Kashmir, and Punjab had B.1.1.7 (alpha variant) as the major lineage, while the Himachal Pradesh state reported B.1.36 as the dominating lineage. A detailed analysis of various domains of S protein was carried out for detecting mutations having a prevalence of >1%; 70, 18, 7, 3, 9, 4, and 1 (N = 112) such mutations were observed in the N-terminal domain, receptor binding domain, C -terminal domain, fusion peptide region, heptapeptide repeat (HR)-1 domains, signal peptide domain, and transmembrane region, respectively. However, no mutations were recorded in the HR-2 and cytoplasmic domains of the S protein. Interestingly, 13.39% (N = 15) of these mutations were reported to increase the infectivity and pathogenicity of the virus; 2% (N = 3) were known to be vaccine breakthrough mutations, and 0.89% (N = 1) were known to escape neutralizing antibodies. The biological significance of 82% (N = 92) of the reported mutations is yet unknown. As SARS-CoV-2 variants are emerging rapidly, it is critical to continuously monitor local viral mutations to understand national trends of virus circulation. This can tremendously help in designing better preventive regimens in the country, and avoid vaccine breakthrough infections.

Analysis of the Neutralizing Activity of Antibodies Targeting Open or Closed SARS-CoV-2 Spike Protein Conformations.

SARS-CoV-2 infection elicits a polyclonal neutralizing antibody (nAb) response that primarily targets the spike protein, but it is still unclear which nAbs are immunodominant and what distinguishes them from subdominant nAbs. This information would however be crucial to predict the evolutionary trajectory of the virus and design future vaccines. To shed light on this issue, we gathered 83 structures of nAbs in complex with spike protein domains. We analyzed in silico the ability of these nAbs to bind the full spike protein trimer in open and closed conformations, and predicted the change in binding affinity of the most frequently observed spike protein variants in the circulating strains. This led us to define four nAb classes with distinct variant escape fractions. By comparing these fractions with those measured from plasma of infected patients, we showed that the class of nAbs that most contributes to the immune response is able to bind the spike protein in its closed conformation. Although this class of nAbs only partially inhibits the spike protein binding to the host's angiotensin converting enzyme 2 (ACE2), it has been suggested to lock the closed pre-fusion spike protein conformation and therefore prevent its transition to an open state. Furthermore, comparison of our predictions with mRNA-1273 vaccinated patient plasma measurements suggests that spike proteins contained in vaccines elicit a different nAb class than the one elicited by natural SARS-CoV-2 infection and suggests the design of highly stable closed-form spike proteins as next-generation vaccine immunogens.

Prediction and Evolution of the Molecular Fitness of SARS-CoV-2 Variants: Introducing SpikePro.

The understanding of the molecular mechanisms driving the fitness of the SARS-CoV-2 virus and its mutational evolution is still a critical issue. We built a simplified computational model, called SpikePro, to predict the SARS-CoV-2 fitness from the amino acid sequence and structure of the spike protein. It contains three contributions: the inter-human transmissibility of the virus predicted from the stability of the spike protein, the infectivity computed in terms of the affinity of the spike protein for the ACE2 receptor, and the ability of the virus to escape from the human immune response based on the binding affinity of the spike protein for a set of neutralizing antibodies. Our model reproduces well the available experimental, epidemiological and clinical data on the impact of variants on the biophysical characteristics of the virus. For example, it is able to identify circulating viral strains that, by increasing their fitness, recently became dominant at the population level. SpikePro is a useful, freely available instrument which predicts rapidly and with good accuracy the dangerousness of new viral strains. It can be integrated and play a fundamental role in the genomic surveillance programs of the SARS-CoV-2 virus that, despite all the efforts, remain time-consuming and expensive.

Chicken Egg Yolk Antibodies (IgYs) block the binding of multiple SARS-CoV-2 spike protein variants to human ACE2.

The SARS-CoV-2 virus is still spreading worldwide, and there is an urgent need to effectively prevent and control this pandemic. This study evaluated the potential efficacy of Egg Yolk Antibodies (IgY) as a neutralizing agent against the SARS-CoV-2. We investigated the neutralizing effect of anti-spike-S1 IgYs on the SARS-CoV-2 pseudovirus, as well as its inhibitory effect on the binding of the coronavirus spike protein mutants to human ACE2. Our results show that the anti-Spike-S1 IgYs showed significant neutralizing potency against SARS-CoV-2 pseudovirus, various spike protein mutants, and even SARS-CoV in vitro. It might be a feasible tool for the prevention and control of ongoing COVID-19.

Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69-V70, France, August to December 2020.

We report the strategy leading to the first detection of variant of concern 202012/01 (VOC) in France (21 December 2020). First, the spike (S) deletion H69-V70 (ΔH69/ΔV70), identified in certain SARS-CoV-2 variants including VOC, is screened for. This deletion is associated with a S-gene target failure (SGTF) in the three-target RT-PCR assay (TaqPath kit). Subsequently, SGTF samples are whole genome sequenced. This approach revealed mutations co-occurring with ΔH69/ΔV70 including S:N501Y in the VOC.