Antibody isotype epitope mapping of SARS-CoV-2 Spike RBD protein: Targets for COVID-19 symptomatology and disease control.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still poses a challenge for biomedicine and public health. To advance the development of effective diagnostic, prognostic, and preventive interventions, our study focused on high-throughput antibody binding epitope mapping of the SARS-CoV-2 spike RBD protein by IgA, IgM and IgG antibodies in saliva and sera of different cohorts from healthy uninfected individuals to SARS-CoV-2-infected unvaccinated and vaccinated asymptomatic, recovered, nonsevere, and severe patients. Identified candidate diagnostic (455-LFRKSNLKPFERD-467), prognostic (395-VYADSFVIRGDEV-407-C-KLH, 332-ITNLCPFGEV-342-C-KLH, 352-AWNRKRI-358-C-KLH, 524-VCGPKKSTNLVKN-536-KLH), and protective (MKLLE-487-NCYFPLQSYGFQPTNGVG-504-GGGGS-446-GGNYNYLYRLFRKSNLKPFERD-467) epitopes were validated with sera from prevaccine and postvaccine cohorts. The results identified neutralizing epitopes and support that antibody recognition of linear B-cell epitopes in RBD protein is associated with antibody isotype and disease symptomatology. The findings in asymptomatic individuals suggest a role for anti-RBD antibodies in the protective response against SARS-CoV-2. The possibility of translating results into diagnostic interventions for the early diagnosis of asymptomatic individuals and prognosis of disease severity provides new tools for COVID-19 surveillance and evaluation of risks in hospitalized patients. These results, together with other approaches, may contribute to the development of new vaccines for the control of COVID-19 and other coronavirus-related diseases using a quantum vaccinomics approach through the combination of protective epitopes.

Linear epitope mapping of the humoral response against SARS-CoV-2 in two independent African cohorts.

Profiling of the antibody responses to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) proteins in African populations is scarce. Here, we performed a detailed IgM and IgG epitope mapping study against 487 peptides covering SARS-CoV-2 wild-type structural proteins. A panel of 41 pre-pandemic and 82 COVID-19 RT-PCR confirmed sera from Madagascar and Senegal were used. We found that the main 36 immunodominant linear epitopes identified were (i) similar in both countries, (ii) distributed mainly in the Spike and the Nucleocapsid proteins, (iii) located outside the RBD and NTD regions where most of the reported SARS-CoV-2 variant mutations occur, and (iv) identical to those reported in European, North American, and Asian studies. Within the severe group, antibody levels were inversely correlated with the viral load. This first antibody epitope mapping study performed in patients from two African countries may be helpful to guide rational peptide-based diagnostic assays or vaccine development.

Characterization and epitope mapping of monoclonal antibodies against PEDV N protein.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea, vomiting, dehydration and high mortality in neonatal piglets. The nucleocapsid (N) protein of PEDV is a highly conserved protein with strong immunogenicity and palys an important role in PEDV diagnosis. However, epitopes on the PEDV N protein have not yet been well characterized. Here, 32 monoclonal antibodies (mAbs) against the PEDV N protein were produced and identified. Six new epitopes were first identified by using a high-throughput epitope mapping method named AbMap. Sequence analysis revealed that among the six epitopes five epitopes were highly conserved among different PEDV strains. We also confirmed that the mAbs derived from the six epitopes of PEDV N protein, have no cross-reactivity with transmissible gastro enteritis virus or porcine delta coronavirus. These mAbs and their defined epitopes will help to understand the N protein structure and immunological characteristics, and to develop a rapid, accurate PEDV diagnosis method.

Antigenicity Alternations of Variant PEDV S Protein Disclosed by Linear B Cell Epitope Mapping.

The spike protein (S) plays a crucial role in porcine epidemic diarrhea virus (PEDV) infection and induces neutralizing antibodies. Mutations of the S protein are supposed to provide the main antigenic shift leading to the antigenic escape of PEDVs. It is therefore a significant question how much accumulation of antigenic shift could lead to the antigenic escape of the variant PEDV. To provide an answer in the study, B cell epitopes (BCEs) on the S protein of the PEDV vaccine strain CV777 (SCV777) and variant strain SD2014 (SSD2014) were mapped using biosynthetic peptides and rabbit anti-PEDV S serum. Seventy-nine and 68 linear BCEs were identified from SCV777 and SSD2014, respectively. While 66.2% of the BCEs of SSD2014 could be recognized by anti-SCV777 serum and 67.1% of SCV777 BCEs could be recognized by anti-SSD2014 serum, more than 40% of the BCEs identified using anti-SCV777 serum on SCV777 could not be recognized by anti-SSD2014 serum and vice versa. The completely shared BCEs took low percentages of 29.4% and 25.3% for SSD2014 and SCV777, respectively. These results indicate a low conservation of antigenicity of the S protein compared to a relatively high amino acid sequence similarity of 92.2% between the two strains. The study provided a BCE shift reference of PEDV antigenic escape and surveillance control.

A framework for predicting variable-length epitopes of human-adapted viruses using machine learning methods.

The coronavirus disease 2019 pandemic has alerted people of the threat caused by viruses. Vaccine is the most effective way to prevent the disease from spreading. The interaction between antibodies and antigens will clear the infectious organisms from the host. Identifying B-cell epitopes is critical in vaccine design, development of disease diagnostics and antibody production. However, traditional experimental methods to determine epitopes are time-consuming and expensive, and the predictive performance using the existing in silico methods is not satisfactory. This paper develops a general framework to predict variable-length linear B-cell epitopes specific for human-adapted viruses with machine learning approaches based on Protvec representation of peptides and physicochemical properties of amino acids. QR decomposition is incorporated during the embedding process that enables our models to handle variable-length sequences. Experimental results on large immune epitope datasets validate that our proposed model's performance is superior to the state-of-the-art methods in terms of AUROC (0.827) and AUPR (0.831) on the testing set. Moreover, sequence analysis also provides the results of the viral category for the corresponding predicted epitopes with high precision. Therefore, this framework is shown to reliably identify linear B-cell epitopes of human-adapted viruses given protein sequences and could provide assistance for potential future pandemics and epidemics.

Severe Acute Respiratory Syndrome Coronavirus 2 Epitope Mapping for Antibodies.

Epitope mapping of the interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Abs is challenging because of complexity in protein three-dimensional structures. Protein structure fingerprint technology was applied for epitope mapping of 44 SARS-CoV-2 Abs with three-dimensional structure complexes. The results defined how the epitopes were distributed on SARS-CoV-2 and how the patterns of six CDRs from Abs participated in neutralization. Also, the residue-residue recognition revealed that certain residues had higher frequencies on the interfaces between SARS-CoV-2 and Abs, and the activity correlated with the physicochemical properties of the residues at the interface. Thus, epitope mapping provides significant lead information for development of epitope-based designs for Abs, vaccines, and diagnostic reagents. This is a bioinformatics project of structural data analysis; no animals or cells were used.

Epitope Mapping of an Anti-Chinese/Golden Hamster Podoplanin Monoclonal Antibody.

Chinese hamster (Cricetulus griseus) and golden hamster (Mesocricetus auratus) are important animal models of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, which affect several organs, including respiratory tract, lung, and kidney. Podoplanin (PDPN) is a marker of lung type I alveolar cells, kidney podocytes, and lymphatic endothelial cells. The development of anti-PDPN monoclonal antibodies (mAbs) for these animals is essential to evaluate the pathogenesis by SARS-CoV-2 infections. Using the Cell-Based Immunization and Screening method, we previously developed an anti-Chinese hamster PDPN (ChamPDPN) mAb, PMab-281 (mouse IgG3, kappa), and further changed its subclass into IgG2a (281-mG2a-f), both of which can recognize not only ChamPDPN but also golden hamster PDPN (GhamPDPN) by flow cytometry and immunohistochemistry. In this study, we examined the critical epitope of 281-mG2a-f, using enzyme-linked immunosorbent assay (ELISA) with synthesized peptides. First, we performed ELISA with peptides derived from ChamPDPN and GhamPDPN extracellular domain, and found that 281-mG2a-f reacted with the peptides, which commonly possess the KIPFEELxT sequence. Next, we analyzed the reaction with the alanine-substituted mutants, and revealed that 281-mG2a-f did not recognize the alanine-substituted peptides of I75A, F77A, and E79A of ChamPDPN. Furthermore, these peptides could not inhibit the recognition of 281-mG2a-f to ChamPDPN-expressing cells by flow cytometry. The results indicate that the binding epitope of 281-mG2a-f includes Ile75, Phe77, and Glu79 of ChamPDPN, which are shared with GhamPDPN.

Development of a Monoclonal Antibody PMab-292 Against Ferret Podoplanin.

Ferrets (Mustela putorius furo) have been used as small animal models to investigate severe acute respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) infections. Pathological analyses of these tissue samples, including those of the lung, are, therefore, essential to understand the pathogenesis of SARS-CoVs and evaluate the action of therapeutic monoclonal antibodies (mAbs) against this disease. However, mAbs that recognize ferret-derived proteins and distinguish between specific cell types, such as lung epithelial cells, are limited. Podoplanin (PDPN) has been identified as an essential marker in lung type I alveolar epithelial cells, kidney podocytes, and lymphatic endothelial cells. In this study, an anti-ferret PDPN (ferPDPN) mAb PMab-292 (mouse IgG1, kappa) was established using the Cell-Based Immunization and Screening (CBIS) method. PMab-292 recognized ferPDPN-overexpressed Chinese hamster ovary-K1 (CHO/ferPDPN) cells by flow cytometry and Western blotting. The kinetic analysis using flow cytometry showed that the KD of PMab-292 for CHO/ferPDPN was 3.4 × 10-8 M. Furthermore, PMab-292 detected lung type I alveolar epithelial cells, lymphatic endothelial cells, and glomerular/Bowman's capsule in the kidney using immunohistochemistry. Hence, these results propose the usefulness of PMab-292 in analyzing ferret-derived tissues for SARS-CoV-2 research.

Epitope mapping of neutralising anti-SARS-CoV-2 monoclonal antibodies: Implications for immunotherapy and vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. This disease has currently affected more than 346 million people and resulted in more than 5.5 million deaths in many countries. Neutralising monoclonal antibodies (MAbs) against the SARS-CoV-2 virus could serve as prophylactic/therapeutic agents in COVID-19 infection by providing passive protection against the virus in individuals. Until now, no Food and Drug Administration/European Medicines Agency-approved neutralising MAb against SARS-CoV-2 virus exists in the market, though a number of MAbs have been authorised for emergency use. Therefore, there is an urgent need for development of efficient anti-SARS-CoV-2 neutralising MAbs for use in the clinic. Moreover, neutralising anti-SARS-CoV-2 MAbs could be used as beneficial tools for designing epitope-based vaccines against the virus. Given that the target epitope of a MAb is a crucial feature influencing its neutralising potency, target epitopes of neutralising anti-SARS-CoV-2 MAbs already reported in the literature and reactivity of these MAbs with SARS-CoV-2 variants are reviewed herein.

Cross-Recognition of SARS-CoV-2 B-Cell Epitopes with Other Betacoronavirus Nucleoproteins.

The B and T lymphocytes of the adaptive immune system are important for the control of most viral infections, including COVID-19. Identification of epitopes recognized by these cells is fundamental for understanding how the immune system detects and removes pathogens, and for antiviral vaccine design. Intriguingly, several cross-reactive T lymphocyte epitopes from SARS-CoV-2 with other betacoronaviruses responsible for the common cold have been identified. In addition, antibodies that cross-recognize the spike protein, but not the nucleoprotein (N protein), from different betacoronavirus have also been reported. Using a consensus of eight bioinformatic methods for predicting B-cell epitopes and the collection of experimentally detected epitopes for SARS-CoV and SARS-CoV-2, we identified four surface-exposed, conserved, and hypothetical antigenic regions that are exclusive of the N protein. These regions were analyzed using ELISA assays with two cohorts: SARS-CoV-2 infected patients and pre-COVID-19 samples. Here we describe four epitopes from SARS-CoV-2 N protein that are recognized by the humoral response from multiple individuals infected with COVID-19, and are conserved in other human coronaviruses. Three of these linear surface-exposed sequences and their peptide homologs in SARS-CoV-2 and HCoV-OC43 were also recognized by antibodies from pre-COVID-19 serum samples, indicating cross-reactivity of antibodies against coronavirus N proteins. Different conserved human coronaviruses (HCoVs) cross-reactive B epitopes against SARS-CoV-2 N protein are detected in a significant fraction of individuals not exposed to this pandemic virus. These results have potential clinical implications.

Limited Recognition of Highly Conserved Regions of SARS-CoV-2.

Understanding the immune response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) is critical to overcome the current coronavirus disease (COVID-19) pandemic. Efforts are being made to understand the potential cross-protective immunity of memory T cells, induced by prior encounters with seasonal coronaviruses, in providing protection against severe COVID-19. In this study we assessed T-cell responses directed against highly conserved regions of SARS-CoV-2. Epitope mapping revealed 16 CD8+ T-cell epitopes across the nucleocapsid (N), spike (S), and open reading frame (ORF)3a proteins of SARS-CoV-2 and five CD8+ T-cell epitopes encoded within the highly conserved regions of the ORF1ab polyprotein of SARS-CoV-2. Comparative sequence analysis showed high conservation of SARS-CoV-2 ORF1ab T-cell epitopes in seasonal coronaviruses. Paradoxically, the immune responses directed against the conserved ORF1ab epitopes were infrequent and subdominant in both convalescent and unexposed participants. This subdominant immune response was consistent with a low abundance of ORF1ab encoded proteins in SARS-CoV-2 infected cells. Overall, these observations suggest that while cross-reactive CD8+ T cells likely exist in unexposed individuals, they are not common and therefore are unlikely to play a significant role in providing broad preexisting immunity in the community. IMPORTANCE T cells play a critical role in protection against SARS-CoV-2. Despite being highly topical, the protective role of preexisting memory CD8+ T cells, induced by prior exposure to circulating common coronavirus strains, remains less clear. In this study, we established a robust approach to specifically assess T cell responses to highly conserved regions within SARS-CoV-2. Consistent with recent observations we demonstrate that recognition of these highly conserved regions is associated with an increased likelihood of milder disease. However, extending these observations we observed that recognition of these conserved regions is rare in both exposed and unexposed volunteers, which we believe is associated with the low abundance of these proteins in SARS-CoV-2 infected cells. These observations have important implications for the likely role preexisting immunity plays in controlling severe disease, further emphasizing the importance of vaccination to generate the immunodominant T cells required for immune protection.

Screening and identification of B cell epitope of the nucleocapsid protein in SARS-CoV-2 using the monoclonal antibodies.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease (COVID-19). It is confirmed that nucleocapsid (N) protein is closely related to viral pathogenesis, modulation of host immune response, RNA transcription, and replication and virus packaging. Therefore, the N protein is a preponderant antigen target for virus detection. The codon-optimized N gene was designed according to the encoding characteristics of insect cells and inserted into pFastBacTM1 vector with 6 × His-tag-fused N protein for expression in insect sf21 cells. Six anti-N mAbs (4G3, 5B3, 12B6, 18C7-A2, 21H10-A3, 21H10-E9) were prepared by recombinant N protein. The mAbs showed high titers, antibody affinity, and reactivity with the SARS-CoV-2 N protein. Then, fourteen overlapped peptides that covered the intact N protein were synthesized (N1-N14). Peptide N14 was identified as the main linear B-cell epitope region via peptide-ELISA and dot-blot assay, and this region was truncated gradually until mapping the peptide 401-DFSKQLQQ-408. Simultaneously, compared with the sequence of variants of concern (VOCs) and variants of interest (VOIs) strains among the several countries, epitope 401-DFSKQLQQ-408 is very conservative among them. The findings provide new guidance for the design and detection of COVID-19 targets. KEY POINTS: • The N protein was optimized according to the insect cell codon preference and was highly expressed. • The monoclonal antibodies prepared in this study were shown high antibody titers and high affinity. • Monoclonal antibodies were used to map the epitope 401-408 amino acids of N protein for the first time in this study.

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.

High-Resolution Linear Epitope Mapping of the Receptor Binding Domain of SARS-CoV-2 Spike Protein in COVID-19 mRNA Vaccine Recipients.

The prompt rollout of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccine is facilitating population immunity, which is becoming more dominant than natural infection-mediated immunity. In the midst of coronavirus disease 2019 (COVID-19) vaccine deployment, understanding the epitope profiles of vaccine-elicited antibodies will be the first step in assessing the functionality of vaccine-induced immunity. In this study, the high-resolution linear epitope profiles of Pfizer-BioNTech COVID-19 mRNA vaccine recipients and COVID-19 patients were delineated by using microarrays mapped with overlapping peptides of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The vaccine-induced antibodies targeting the RBD had a broader distribution across the RBD than that induced by the natural infection. Half-maximal neutralization titers were measured in vitro by live virus neutralization assays. As a result, relatively lower neutralizability was observed in vaccine recipient sera, when normalized to a total anti-RBD IgG titer. However, mutation panel assays targeting the SARS-CoV-2 variants of concern have shown that the vaccine-induced epitope variety, rich in breadth, may grant resistance against future viral evolutionary escapes, serving as an advantage of vaccine-induced immunity. IMPORTANCE Establishing vaccine-based population immunity has been the key factor in attaining herd protection. Thanks to expedited worldwide research efforts, the potency of mRNA vaccines against the coronavirus disease 2019 (COVID-19) is now incontestable. The next debate is regarding the coverage of SARS-CoV-2 variants. In the midst of vaccine deployment, it is of importance to describe the similarities and differences between the immune responses of COVID-19 vaccine recipients and naturally infected individuals. In this study, we demonstrated that the antibody profiles of vaccine recipients are richer in variety, targeting a key protein of the invading virus, than those of naturally infected individuals. Vaccine-elicited antibodies included more nonneutralizing antibodies than infection-elicited antibodies, and their breadth in antibody variations suggested possible resilience against future SARS-CoV-2 variants. The antibody profile achieved by vaccinations in naive individuals provides important insight into the first step toward vaccine-based population immunity.

Mapping cross-variant neutralizing sites on the SARS-CoV-2 spike protein.

The emergence of multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern threatens the efficacy of currently approved vaccines and authorized therapeutic monoclonal antibodies (MAbs). It is hence important to continue searching for SARS-CoV-2 broadly neutralizing MAbs and defining their epitopes. Here, we isolate 9 neutralizing mouse MAbs raised against the spike protein of a SARS-CoV-2 prototype strain and evaluate their neutralizing potency towards a panel of variants, including B.1.1.7, B.1.351, B.1.617.1, and B.1.617.2. By using a combination of biochemical, virological, and cryo-EM structural analyses, we identify three types of cross-variant neutralizing MAbs, represented by S5D2, S5G2, and S3H3, respectively, and further define their epitopes. S5D2 binds the top lateral edge of the receptor-binding motif within the receptor-binding domain (RBD) with a binding footprint centred around the loop477-489, and efficiently neutralizes all variant pseudoviruses, but the potency against B.1.617.2 was observed to decrease significantly. S5G2 targets the highly conserved RBD core region and exhibits comparable neutralization towards the variant panel. S3H3 binds a previously unreported epitope located within the evolutionarily stable SD1 region and is able to near equally neutralize all of the variants tested. Our work thus defines three distinct cross-variant neutralizing sites on the SARS-CoV-2 spike protein, providing guidance for design and development of broadly effective vaccines and MAb-based therapies.

Detailed epitope mapping of SARS-CoV-2 nucleoprotein reveals specific immunoresponse in cats and dogs housed with COVID-19 patients.

Since the initial emergence in December 2019, the novel Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been reported in over 200 countries, representing an unprecedented challenge related to disease control worldwide. In this context, cases of human to animal transmission have been reported, raising concern about the potential role of companion animals in the pandemic and stressing the need for reliable animal testing. In the study, a detailed epitope mapping of SARS-CoV-2 nucleoprotein, using both human and pet sera, allowed the identification of the most antigenic region in the C-terminus domain of the protein, which was used to develop an experimental double antigen-based ELISA. A panel of pre-pandemic sera and sera of animals immunized against (or naturally infected with) related coronaviruses was used to assess assay specificity at 99.5%. Positive sera belonging to animals housed with COVID-19 patients were confirmed with the experimental double-antigen ELISA using Plaque Reduction Neutralization test (PRNT) test as gold standard. The availability of a serological assay that targets a highly specific viral antigen represents a valuable tool for multispecies monitoring of Coronavirus Disease 2019 (COVID-19) infection in susceptible animals.

High-resolution epitope mapping and characterization of SARS-CoV-2 antibodies in large cohorts of subjects with COVID-19.

As Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to spread, characterization of its antibody epitopes, emerging strains, related coronaviruses, and even the human proteome in naturally infected patients can guide the development of effective vaccines and therapies. Since traditional epitope identification tools are dependent upon pre-defined peptide sequences, they are not readily adaptable to diverse viral proteomes. The Serum Epitope Repertoire Analysis (SERA) platform leverages a high diversity random bacterial display library to identify proteome-independent epitope binding specificities which are then analyzed in the context of organisms of interest. When evaluating immune response in the context of SARS-CoV-2, we identify dominant epitope regions and motifs which demonstrate potential to classify mild from severe disease and relate to neutralization activity. We highlight SARS-CoV-2 epitopes that are cross-reactive with other coronaviruses and demonstrate decreased epitope signal for mutant SARS-CoV-2 strains. Collectively, the evolution of SARS-CoV-2 mutants towards reduced antibody response highlight the importance of data-driven development of the vaccines and therapies to treat COVID-19.

Immunoinformatics mapping of potential epitopes in SARS-CoV-2 structural proteins.

All approved coronavirus disease 2019 (COVID-19) vaccines in current use are safe, effective, and reduce the risk of severe illness. Although data on the immunological presentation of patients with COVID-19 is limited, increasing experimental evidence supports the significant contribution of B and T cells towards the resolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Despite the availability of several COVID-19 vaccines with high efficacy, more effective vaccines are still needed to protect against the new variants of SARS-CoV-2. Employing a comprehensive immunoinformatic prediction algorithm and leveraging the genetic closeness with SARS-CoV, we have predicted potential immune epitopes in the structural proteins of SARS-CoV-2. The S and N proteins of SARS-CoV-2 and SARS-CoVs are main targets of antibody detection and have motivated us to design four multi-epitope vaccines which were based on our predicted B- and T-cell epitopes of SARS-CoV-2 structural proteins. The cardinal epitopes selected for the vaccine constructs are predicted to possess antigenic, non-allergenic, and cytokine-inducing properties. Additionally, some of the predicted epitopes have been experimentally validated in published papers. Furthermore, we used the C-ImmSim server to predict effective immune responses induced by the epitope-based vaccines. Taken together, the immune epitopes predicted in this study provide a platform for future experimental validations which may facilitate the development of effective vaccine candidates and epitope-based serological diagnostic assays.

In Silico Designing of a Multitope Vaccine against Rhizopus microsporus with Potential Activity against Other Mucormycosis Causing Fungi.

During the current era of the COVID-19 pandemic, the dissemination of Mucorales has been reported globally, with elevated rates of infection in India, and because of the high rate of mortality and morbidity, designing an effective vaccine against mucormycosis is a major health priority, especially for immunocompromised patients. In the current study, we studied shared Mucorales proteins, which have been reported as virulence factors, and after analysis of several virulent proteins for their antigenicity and subcellular localization, we selected spore coat (CotH) and serine protease (SP) proteins as the targets of epitope mapping. The current study proposes a vaccine constructed based on top-ranking cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B cell lymphocyte (BCL) epitopes from filtered proteins. In addition to the selected epitopes, ß-defensins adjuvant and PADRE peptide were included in the constructed vaccine to improve the stimulated immune response. Computational tools were used to estimate the physicochemical and immunological features of the proposed vaccine and validate its binding with TLR-2, where the output data of these assessments potentiate the probability of the constructed vaccine to stimulate a specific immune response against mucormycosis. Here, we demonstrate the approach of potential vaccine construction and assessment through computational tools, and to the best of our knowledge, this is the first study of a proposed vaccine against mucormycosis based on the immunoinformatics approach.

Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations.

Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.