Multi-pathogen based chimeric vaccine to fight against COVID-19 and concomitant coinfections.

BACKGROUND: COVID-19 has proved to be a fatal disease of the year 2020, due to which thousands of people globally have lost their lives, and still, the infection cases are at a high rate. Experimental studies suggested that SARS-CoV-2 interacts with various microorganisms, and this coinfection is accountable for the augmentation of infection severity. METHODS AND RESULTS: In this study, we have designed a multi-pathogen vaccine by involving the immunogenic proteins from S. pneumonia, H. influenza, and M. tuberculosis, as they are dominantly associated with SARS-CoV-2. A total of 8 antigenic protein sequences were selected to predict B-cell, HTL, and CTL epitopes restricted to the most prevalent HLA alleles. The selected epitopes were antigenic, non-allergenic, and non-toxic and were linked with adjuvant and linkers to make the vaccine protein more immunogenic, stable, and flexible. The tertiary structure, Ramachandran plot, and discontinuous B-cell epitopes were predicted. Docking and MD simulation study has shown efficient binding of the chimeric vaccine with the TLR4 receptor. CONCLUSION: The in silico immune simulation analysis has shown a high level of cytokines and IgG after a three-dose injection. Hence, this strategy could be a better way to decrease the disease's severity and could be used as a weapon to prevent this pandemic.

Identification of B-Cell Linear Epitopes in the Nucleocapsid (N) Protein B-Cell Linear Epitopes Conserved among the Main SARS-CoV-2 Variants.

The Nucleocapsid (N) protein is highlighted as the main target for COVID-19 diagnosis by antigen detection due to its abundance in circulation early during infection. However, the effects of the described mutations in the N protein epitopes and the efficacy of antigen testing across SARS-CoV-2 variants remain controversial and poorly understood. Here, we used immunoinformatics to identify five epitopes in the SARS-CoV-2 N protein (N(34-48), N(89-104), N(185-197), N(277-287), and N(378-390)) and validate their reactivity against samples from COVID-19 convalescent patients. All identified epitopes are fully conserved in the main SARS-CoV-2 variants and highly conserved with SARS-CoV. Moreover, the epitopes N(185-197) and N(277-287) are highly conserved with MERS-CoV, while the epitopes N(34-48), N(89-104), N(277-287), and N(378-390) are lowly conserved with common cold coronaviruses (229E, NL63, OC43, HKU1). These data are in accordance with the observed conservation of amino acids recognized by the antibodies 7R98, 7N0R, and 7CR5, which are conserved in the SARS-CoV-2 variants, SARS-CoV and MERS-CoV but lowly conserved in common cold coronaviruses. Therefore, we support the antigen tests as a scalable solution for the population-level diagnosis of SARS-CoV-2, but we highlight the need to verify the cross-reactivity of these tests against the common cold coronaviruses.

SARS-CoV-2 Omicron boosting induces de novo B cell response in humans.

The primary two-dose SARS-CoV-2 mRNA vaccine series are strongly immunogenic in humans, but the emergence of highly infectious variants necessitated additional doses and the development of vaccines aimed at the new variants1-4. SARS-CoV-2 booster immunizations in humans primarily recruit pre-existing memory B cells5-9. However, it remains unclear whether the additional doses induce germinal centre reactions whereby re-engaged B cells can further mature, and whether variant-derived vaccines can elicit responses to variant-specific epitopes. Here we show that boosting with an mRNA vaccine against the original monovalent SARS-CoV-2 mRNA vaccine or the bivalent B.1.351 and B.1.617.2 (Beta/Delta) mRNA vaccine induced robust spike-specific germinal centre B cell responses in humans. The germinal centre response persisted for at least eight weeks, leading to significantly more mutated antigen-specific bone marrow plasma cell and memory B cell compartments. Spike-binding monoclonal antibodies derived from memory B cells isolated from individuals boosted with either the original SARS-CoV-2 spike protein, bivalent Beta/Delta vaccine or a monovalent Omicron BA.1-based vaccine predominantly recognized the original SARS-CoV-2 spike protein. Nonetheless, using a more targeted sorting approach, we isolated monoclonal antibodies that recognized the BA.1 spike protein but not the original SARS-CoV-2 spike protein from individuals who received the mRNA-1273.529 booster; these antibodies were less mutated and recognized novel epitopes within the spike protein, suggesting that they originated from naive B cells. Thus, SARS-CoV-2 booster immunizations in humans induce robust germinal centre B cell responses and can generate de novo B cell responses targeting variant-specific epitopes.

Design of a multi-epitope-based vaccine consisted of immunodominant epitopes of structural proteins of SARS-CoV-2 using immunoinformatics approach.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown rapid global spread and has resulted in a significant death toll worldwide. In this study, we aimed to design a multi-epitope vaccine against SARS-CoV-2 based on structural proteins S, M, N, and E. We identified B- and T-cell epitopes and then the antigenicity, toxicity, allergenicity, and similarity of predicted epitopes were analyzed. T-cell epitopes were docked with corresponding HLA alleles. Consequently, the selected T- and B-cell epitopes were included in the final construct. All selected epitopes were connected with different linkers and flagellin and pan-HLA DR binding epitopes (PADRE) as an adjuvant were used in the vaccine construct. Furthermore, molecular docking was used to evaluate the complex between the final vaccine construct and two alleles, HLA-A*02:01 and HLA-DRB1*01:01. Finally, codons were optimized for in silico cloning into pET28a(+) vector using SnapGene. The final vaccine construct comprised 11 CTL, HTL, and B-cell epitopes corresponding to 394 amino acid residues. In silico evaluation showed that the designed vaccine might potentially promote an immune response. Further in vivo preclinical and clinical testing is required to determine the safety and efficacy of the designed vaccine.

Identification of a novel linear B-cell epitope in porcine deltacoronavirus nucleocapsid protein.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that caused diarrhea and/or vomiting in neonatal piglets worldwide. Coronaviruses nucleocapsid (N) protein is the most conserved structural protein for viral replication and possesses good antigenicity. In this study, three monoclonal antibodies (mAbs), 3B4, 4D3, and 4E3 identified as subclass IgG2aκ were prepared using the lymphocytic hybridoma technology against PDCoV N protein. Furthermore, the B-cell epitope recognized by mAb 4D3 was mapped by dozens of overlapping truncated recombinant proteins based on the western blotting. The polypeptide 28QFRGNGVPLNSAIKPVE44 (EP-4D3) in the N-terminal of PDCoV N protein was identified as the minimal linear epitope for binding mAb 4D3. And the EP-4D3 epitope's amino acid sequence homology study revealed that PDCoV strains are substantially conserved, with the exception of the Alanine43 substitution Valine43 in the China lineage, the Early China lineage, and the Thailand, Vietnam, and Laos lineage. The epitope sequences shared high similarity (94.1%) with porcine coronavirus HKU15-155 (PorCoV HKU15), Asian leopard cats coronavirus (ALCCoV), sparrow coronavirus HKU17 (SpCoV HKU17), and sparrow deltacoronavirus. In contrast, the epitope sequences shared a very low homology (11.8 to 29.4%) with other porcine CoVs (PEDV, TGEV, PRCV, SADS-CoV, PHEV). Overall, the study will enrich the biological function of PDCoV N protein and provide foundational data for further development of diagnostic applications. KEY POINTS: • Three monoclonal antibodies against PDCoV N protein were prepared. • Discovery of a novel B-cell liner epitope (28QFRGNGVPLNSAIKPVE44) of PDCoV N protein. • The epitope EP-4D3 was conserved among PDCoV strains.

Bioinformatics-based SARS-CoV-2 epitopes design and the impact of spike protein mutants on epitope humoral immunities.

BACKGROUND: Epitope selection is the key to peptide vaccines development. Bioinformatics tools can efficiently improve the screening of antigenic epitopes and help to choose the right ones. OBJECTIVE: To predict, synthesize and testify peptide epitopes at spike protein, assess the effect of mutations on epitope humoral immunity, thus provide clues for the design and development of epitope peptide vaccines against SARS-CoV-2. METHODS: Bioinformatics servers and immunological tools were used to identify the helper T lymphocyte, cytotoxic T lymphocyte, and linear B lymphocyte epitopes on the S protein of SARS-CoV-2. Physicochemical properties of candidate epitopes were analyzed using IEDB, VaxiJen, and AllerTOP online software. Three candidate epitopes were synthesized and their antigenic responses were evaluated by binding antibody detection. RESULTS: A total of 20 antigenic, non-toxic and non-allergenic candidate epitopes were identified from 1502 epitopes, including 6 helper T-cell epitopes, 13 cytotoxic T-cell epitopes, and 1 linear B cell epitope. After immunization with antigen containing candidate epitopes S206-221, S403-425, and S1157-1170 in rabbits, the binding titers of serum antibody to the corresponding peptide, S protein, receptor-binding domain protein were (415044, 2582, 209.3), (852819, 45238, 457767) and (357897, 10528, 13.79), respectively. The binding titers to Omicron S protein were 642, 12,878 and 7750, respectively, showing that N211L, DEL212 and K417N mutations cause the reduction of the antibody binding activity. CONCLUSIONS: Bioinformatic methods are effective in peptide epitopes design. Certain mutations of the Omicron would lead to the loss of antibody affinity to Omicron S protein.

Using Self-Assembling ADDomer Platform to Display B and T Epitopes of Type O Foot-and-Mouth Disease Virus.

Foot-and-mouth disease virus (FMDV) is a highly contagious and devastating virus that infects cloven-hoofed livestock and various wildlife species. Vaccination is the best measure to prevent FMD. ADDomer, as a kind of non-infectious adenovirus-inspired nanoparticle, has the advantage of high thermal stability. In this study, two dominant B-cell antigen epitopes (residues 129~160 and 200~213) and a dominant T-cell antigen epitope (residues 16~44) of type O FMDV were inserted into the ADDomer variable loop (VL) and arginine-glycine-aspartic acid (RGD) loop. The 3D structure of the recombinant protein (ADDomer-RBT) was simulated by homology modeling. First, the recombinant proteins were expressed by the baculovirus expression system and detected by western blot and Q Exactive mass spectrometry. Then the formation of VLPs was observed under a transmission electron micrograph (TEM). Finally, we evaluated the immunogenicity of chimeric VLPs with a murine model. Bioinformatic software analysis preliminarily corroborated that the chosen epitopes were successfully exposed on the surface of ADDomer VLPs. The TEM assay demonstrated the structural integrity of the VLPs. After immunizing, it was found that FMDV-specific antibodies can be produced in mice to induce humoral and cellular immune responses. To sum up, the ADDomer platform can be used as an effective antigen carrier to deliver antigen epitopes. This study presents one of the candidate vaccines to prevent and control FMDV.

Molecular modelling on multiepitope-based vaccine against SARS-CoV-2 using immunoinformatics, molecular docking, and molecular dynamics simulation.

The pandemic of COVID-19 caused by SARS-CoV-2 has made a worldwide health emergency. Despite the fact that current vaccines are readily available, several SARSCoV-2 variants affecting the existing vaccine are to be less effective due to the mutations in the structural proteins. Furthermore, the appearance of the new variants cannot be easily predicted in the future. Therefore, the attempts to construct new vaccines or to modify the current vaccines are still pivotal works for preventing the spread of the virus. In the present investigation, the computational analysis through immunoinformatics, molecular docking, and molecular dynamics (MD) simulation is employed to construct an effective vaccine against SARS-CoV2. The structural proteins of SARS-CoV2 are utilized to create a multiepitope-based vaccine (MEV). According to our findings presented by systematic procedures in the current investigation, the MEV construct may be able to trigger a strong immunological response against the virus. Therefore, the designed MEV could be a potential vaccine candidate against SARS-CoV-2, and also it is expected to be effective for other variants.

Considering epitopes conservity in targeting SARS-CoV-2 mutations in variants: a novel immunoinformatics approach to vaccine design.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has gained mutations at an alarming rate in the past years. Developing mutations can increase the virus's pathogenicity and virulence; reduce the efficacy of vaccines, antibodies neutralization, and even challenge adaptive immunity. So, it is essential to identify conserved epitopes (with fewer mutations) in different variants with appropriate antigenicity to target the variants by an appropriate vaccine design. Yet as, 3369 SARS-CoV-2 genomes were collected from global initiative on sharing avian flu data. Then, mutations in the immunodominant regions (IDRs), immune epitope database (IEDB) epitopes, and also predicted epitopes were calculated. In the following, epitopes conservity score against the total number of events (mutations) and the number of mutated sites in each epitope was weighted by Shannon entropy and then calculated by the Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS). Based on the TOPSIS conservity score and antigenicity score, the epitopes were plotted. The result demonstrates that almost all epitopes and IDRs with various lengths have gained different numbers of mutations in dissimilar sites. Herein, our two-step calculation for conservity recommends only 8 IDRs, 14 IEDB epitopes, and 10 predicted epitopes among all epitopes. The selected ones have higher conservity and higher immunogenicity. This method is an open-source multi-criteria decision-making platform, which provides a scientific approach to selecting epitopes with appropriate conservity and immunogenicity; against ever-changing viruses.

Identification of three conserved linear B cell epitopes on the SARS-CoV-2 spike protein.

Spike (S) glycoprotein is the most significant structural protein of SARS-CoV-2 and a key target for neutralizing antibodies. In light of the on-going SARS-CoV-2 pandemic, identification and screening of epitopes of spike glycoproteins will provide vital progress in the development of sensitive and specific diagnostic tools. In the present study, NTD, RBD, and S2 genes were inserted into the pcDNA3.1(+) vector and designed with N-terminal 6× His-tag for fusion expression in HEK293F cells by transient transfection. Six monoclonal antibodies (4G, 9E, 4B, 7D, 8F, and 3D) were prepared using the expressed proteins by cell fusion technique. The characterization of mAbs was performed by indirect -ELISA, western blot, and IFA. We designed 49 overlapping synthesized peptides that cover the extracellular region of S protein in which 6 amino acid residues were offset between adjacent (S1-S49). Peptides S12, S19, and S49 were identified as the immunodominant epitope regions by the mAbs. These regions were further truncated and the peptides S12.2 286TDAVDCALDPLS297, S19.2 464FERDISTEIYQA475, and S49.4 1202ELGKYEQYIKWP1213 were identified as B- cell linear epitopes for the first time. Alanine scans showed that the D467, I468, E471, Q474, and A475 of the epitope S19.2 and K1205, Q1208, and Y1209 of the epitope S49.4 were the core sites involved in the mAbs binding. The multiple sequence alignment analysis showed that these three epitopes were highly conserved among the variants of concern (VOCs) and variants of interest (VOIs). Taken together, the findings provide a potential material for rapid diagnosis methods of COVID-19.

BA.2.12.1, BA.4 and BA.5 escape antibodies elicited by Omicron infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages BA.2.12.1, BA.4 and BA.5 exhibit higher transmissibility than the BA.2 lineage1. The receptor binding and immune-evasion capability of these recently emerged variants require immediate investigation. Here, coupled with structural comparisons of the spike proteins, we show that BA.2.12.1, BA.4 and BA.5 (BA.4 and BA.5 are hereafter referred collectively to as BA.4/BA.5) exhibit similar binding affinities to BA.2 for the angiotensin-converting enzyme 2 (ACE2) receptor. Of note, BA.2.12.1 and BA.4/BA.5 display increased evasion of neutralizing antibodies compared with BA.2 against plasma from triple-vaccinated individuals or from individuals who developed a BA.1 infection after vaccination. To delineate the underlying antibody-evasion mechanism, we determined the escape mutation profiles2, epitope distribution3 and Omicron-neutralization efficiency of 1,640 neutralizing antibodies directed against the receptor-binding domain of the viral spike protein, including 614 antibodies isolated from people who had recovered from BA.1 infection. BA.1 infection after vaccination predominantly recalls humoral immune memory directed against ancestral (hereafter referred to as wild-type (WT)) SARS-CoV-2 spike protein. The resulting elicited antibodies could neutralize both WT SARS-CoV-2 and BA.1 and are enriched on epitopes on spike that do not bind ACE2. However, most of these cross-reactive neutralizing antibodies are evaded by spike mutants L452Q, L452R and F486V. BA.1 infection can also induce new clones of BA.1-specific antibodies that potently neutralize BA.1. Nevertheless, these neutralizing antibodies are largely evaded by BA.2 and BA.4/BA.5 owing to D405N and F486V mutations, and react weakly to pre-Omicron variants, exhibiting narrow neutralization breadths. The therapeutic neutralizing antibodies bebtelovimab4 and cilgavimab5 can effectively neutralize BA.2.12.1 and BA.4/BA.5, whereas the S371F, D405N and R408S mutations undermine most broadly sarbecovirus-neutralizing antibodies. Together, our results indicate that Omicron may evolve mutations to evade the humoral immunity elicited by BA.1 infection, suggesting that BA.1-derived vaccine boosters may not achieve broad-spectrum protection against new Omicron variants.

Potential linear B-cells epitope change to a helix structure in the spike of Omicron 21L or BA.2 predicts increased SARS-CoV-2 antibodies evasion.

The world health organization has announced that SARS-CoV-2 Omicron variant (B.1.1.529), including the three versions; 21K (BA.1), 21L (BA.2) and 21M (BA.3) as a variant of concern (VOC) on November 2022. In this study, we used the specialized computational platforms to predict the stability and flexibility of the spike protein of Omicron. The aim of this study was to investigate the expected effect of Omicron spike mutations on its physiochemical properties. Findings of this study revealed 16 stabilizing mutations that might explain a newly gained environmental stability. We expect the new mutations to play a crucial role in changing the physiochemical properties of epitopes of the spike protein. The notable finding of SuerPose work was the potential linear B-cells epitope G252 → S255 that has been changed in the spike protein of the Omicron 21L to a helix structure which might confer an escape from human monoclonal antibodies.

In silico SARS-CoV-2 vaccine development for Omicron strain using reverse vaccinology.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic began in 2019 but it remains as a serious threat today. To reduce and prevent spread of the virus, multiple vaccines have been developed. Despite the efforts in developing vaccines, Omicron strain of the virus has recently been designated as a variant of concern (VOC) by the World Health Organization (WHO). OBJECTIVE: To develop a vaccine candidate against Omicron strain (B.1.1.529, BA.1) of the SARS-CoV-19. METHODS: We applied reverse vaccinology methods for BA.1 and BA.2 as the vaccine target and a control, respectively. First, we predicted MHC I, MHC II and B cell epitopes based on their viral genome sequences. Second, after estimation of antigenicity, allergenicity and toxicity, a vaccine construct was assembled and tested for physicochemical properties and solubility. Third, AlphaFold2, RaptorX and RoseTTAfold servers were used to predict secondary structures and 3D structures of the vaccine construct. Fourth, molecular docking analysis was performed to test binding of our construct with angiotensin converting enzyme 2 (ACE2). Lastly, we compared mutation profiles on the epitopes between BA.1, BA.2, and wild type to estimate the efficacy of the vaccine. RESULTS: We collected a total of 10 MHC I, 9 MHC II and 5 B cell epitopes for the final vaccine construct for Omicron strain. All epitopes were predicted to be antigenic, non-allergenic and non-toxic. The construct was estimated to have proper stability and solubility. The best modelled tertiary structures were selected for molecular docking analysis with ACE2 receptor. CONCLUSIONS: These results suggest the potential efficacy of our newly developed vaccine construct as a novel vaccine candidate against Omicron strain of the coronavirus.

Design of a Recombinant Multivalent Epitope Vaccine Based on SARS-CoV-2 and Its Variants in Immunoinformatics Approaches.

The development of an effective multivalent vaccine against SARS-CoV-2 variants is an important means to improve the global public health situation caused by COVID-19. In this study, we identified the antigen epitopes of the main global epidemic SARS-CoV-2 and mutated virus strains using immunoinformatics approach, and screened out 8 cytotoxic T lymphocyte epitopes (CTLEs), 17 helper T lymphocyte epitopes (HTLEs), 9 linear B-cell epitopes (LBEs) and 4 conformational B-cell epitopes (CBEs). The global population coverage of CTLEs and HTLEs was 93.16% and 99.9% respectively. These epitopes were spliced together by corresponding linkers and recombined into multivalent vaccine. In silico tests, the vaccine protein was a non-allergen and the docking with TLR-3 molecule showed a strong interaction. The results of immune simulation showed that the vaccine may be helpful to initiate both cellular and humoral immunity against all VOC. The optimistic immunogenicity of the vaccine was confirmed in vivo and in vitro finally. Therefore, our vaccine may have potential protection against SARS-CoV-2 and its variants.

Reverse vaccinology approach for multi-epitope centered vaccine design against delta variant of the SARS-CoV-2.

The ongoing COVID-19 outbreak, initially identified in Wuhan, China, has impacted people all over the globe and new variants of concern continue to threaten hundreds of thousands of people. The delta variant (first reported in India) is currently classified as one of the most contagious variants of SARS-CoV-2. It is estimated that the transmission rate of delta variant is 225% times faster than the alpha variant, and it is causing havoc worldwide (especially in the USA, UK, and South Asia). The mutations found in the spike protein of delta variant make it more infective than other variants in addition to ruining the global efficacy of available vaccines. In the current study, an in silico reverse vaccinology approach was applied for multi-epitope vaccine construction against the spike protein of delta variant, which could induce an immune response against COVID-19 infection. Non-toxic, highly conserved, non-allergenic and highly antigenic B-cell, HTL, and CTL epitopes were identified to minimize adverse effects and maximize the efficacy of chimeric vaccines that could be developed from these epitopes. Finally, V1 vaccine construct model was shortlisted and 3D modeling was performed by refinement, docking against HLAs and TLR4 protein, simulation and in silico expression. In silico evaluation showed that the designed chimeric vaccine could elicit an immune response (i.e., cell-mediated and humoral) identified through immune simulation. This study could add to the efforts of overcoming global burden of COVID-19 particularly the variants of concern.

Full-genome sequences of the first two SARS-CoV-2 viruses from India.

Background & objectives: Since December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has globally affected 195 countries. In India, suspected cases were screened for SARS-CoV-2 as per the advisory of the Ministry of Health and Family Welfare. The objective of this study was to characterize SARS-CoV-2 sequences from three identified positive cases as on February 29, 2020. Methods: Throat swab/nasal swab specimens for a total of 881 suspected cases were screened by E gene and confirmed by RdRp (1), RdRp (2) and N gene real-time reverse transcription-polymerase chain reactions and next-generation sequencing. Phylogenetic analysis, molecular characterization and prediction of B- and T-cell epitopes for Indian SARS-CoV-2 sequences were undertaken. Results: Three cases with a travel history from Wuhan, China, were confirmed positive for SARS-CoV-2. Almost complete (29,851 nucleotides) genomes of case 1, case 3 and a fragmented genome for case 2 were obtained. The sequences of Indian SARS-CoV-2 though not identical showed high (~99.98%) identity with Wuhan seafood market pneumonia virus (accession number: NC 045512). Phylogenetic analysis showed that the Indian sequences belonged to different clusters. Predicted linear B-cell epitopes were found to be concentrated in the S1 domain of spike protein, and a conformational epitope was identified in the receptor-binding domain. The predicted T-cell epitopes showed broad human leucocyte antigen allele coverage of A and B supertypes predominant in the Indian population. Interpretation & conclusions: The two SARS-CoV-2 sequences obtained from India represent two different introductions into the country. The genetic heterogeneity is as noted globally. The identified B- and T-cell epitopes may be considered suitable for future experiments towards the design of vaccines and diagnostics. Continuous monitoring and analysis of the sequences of new cases from India and the other affected countries would be vital to understand the genetic evolution and rates of substitution of the SARS-CoV-2.

Epitope profiling using computational structural modelling demonstrated on coronavirus-binding antibodies.

Identifying the epitope of an antibody is a key step in understanding its function and its potential as a therapeutic. Sequence-based clonal clustering can identify antibodies with similar epitope complementarity, however, antibodies from markedly different lineages but with similar structures can engage the same epitope. We describe a novel computational method for epitope profiling based on structural modelling and clustering. Using the method, we demonstrate that sequence dissimilar but functionally similar antibodies can be found across the Coronavirus Antibody Database, with high accuracy (92% of antibodies in multiple-occupancy structural clusters bind to consistent domains). Our approach functionally links antibodies with distinct genetic lineages, species origins, and coronavirus specificities. This indicates greater convergence exists in the immune responses to coronaviruses than is suggested by sequence-based approaches. Our results show that applying structural analytics to large class-specific antibody databases will enable high confidence structure-function relationships to be drawn, yielding new opportunities to identify functional convergence hitherto missed by sequence-only analysis.

A vaccine built from potential immunogenic pieces derived from the SARS-CoV-2 spike glycoprotein: A computational approximation.

Coronavirus Disease 2019 (COVID-19) represents a new global threat demanding a multidisciplinary effort to fight its etiological agent-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this regard, immunoinformatics may aid to predict prominent immunogenic regions from critical SARS-CoV-2 structural proteins, such as the spike (S) glycoprotein, for their use in prophylactic or therapeutic interventions against this highly pathogenic betacoronavirus. Accordingly, in this study, an integrated immunoinformatics approach was applied to identify cytotoxic T cell (CTC), T helper cell (THC), and Linear B cell (BC) epitopes from the S glycoprotein in an attempt to design a high-quality multi-epitope vaccine. The best CTC, THC, and BC epitopes showed high viral antigenicity and lack of allergenic or toxic residues, as well as CTC and THC epitopes showed suitable interactions with HLA class I (HLA-I) and HLA class II (HLA-II) molecules, respectively. Remarkably, SARS-CoV-2 receptor-binding domain (RBD) and its receptor-binding motif (RBM) harbour several potential epitopes. The structure prediction, refinement, and validation data indicate that the multi-epitope vaccine has an appropriate conformation and stability. Four conformational epitopes and an efficient binding between Toll-like receptor 4 (TLR4) and the vaccine model were observed. Importantly, the population coverage analysis showed that the multi-epitope vaccine could be used globally. Notably, computer-based simulations suggest that the vaccine model has a robust potential to evoke and maximize both immune effector responses and immunological memory to SARS-CoV-2. Further research is needed to accomplish with the mandatory international guidelines for human vaccine formulations.

Bioengineering of Novel Non-Replicating mRNA (NRM) and Self-Amplifying mRNA (SAM) Vaccine Candidates Against SARS-CoV-2 Using Immunoinformatics Approach.

Presently, the world needs safe and effective vaccines to overcome the COVID-19 pandemic. Our work has focused on formulating two types of mRNA vaccines that differ in capacity to copy themselves inside the cell. These are non-amplifying mRNA (NRM) and self-amplifying mRNA (SAM) vaccines. Both the vaccine candidates encode an engineered viral replicon which can provoke an immune response. Hence we predicted and screened twelve epitopes from the spike glycoprotein of SARS-CoV-2. We used five CTL, four HTL, and three B-cell-activating epitopes to formulate each mRNA vaccine. Molecular docking revealed that these epitopes could combine with HLA molecules that are important for boosting immunogenicity. The B-cell epitopes were adjoined with GPGPG linkers, while CTL and HTL epitopes were linked with KK linkers. The entire protein chain was reverse translated to develop a specific NRM-based vaccine. We incorporate gene encoding replicase in the upstream region of CDS encoding antigen to design the SAM vaccine. Subsequently, signal sequences were added to human mRNA to formulate vaccines. Both vaccine formulations translated to produce the epitopes in host cells, initiate a protective immune cascade, and generate immunogenic memory, which can counter future SARS-CoV-2 viral exposures before the onset of infection.

Viral surface geometry shapes influenza and coronavirus spike evolution through antibody pressure.

The evolution of circulating viruses is shaped by their need to evade antibody response, which mainly targets the viral spike. Because of the high density of spikes on the viral surface, not all antigenic sites are targeted equally by antibodies. We offer here a geometry-based approach to predict and rank the probability of surface residues of SARS spike (S protein) and influenza H1N1 spike (hemagglutinin) to acquire antibody-escaping mutations utilizing in-silico models of viral structure. We used coarse-grained MD simulations to estimate the on-rate (targeting) of an antibody model to surface residues of the spike protein. Analyzing publicly available sequences, we found that spike surface sequence diversity of the pre-pandemic seasonal influenza H1N1 and the sarbecovirus subgenus highly correlates with our model prediction of antibody targeting. In particular, we identified an antibody-targeting gradient, which matches a mutability gradient along the main axis of the spike. This identifies the role of viral surface geometry in shaping the evolution of circulating viruses. For the 2009 H1N1 and SARS-CoV-2 pandemics, a mutability gradient along the main axis of the spike was not observed. Our model further allowed us to identify key residues of the SARS-CoV-2 spike at which antibody escape mutations have now occurred. Therefore, it can inform of the likely functional role of observed mutations and predict at which residues antibody-escaping mutation might arise.