Repeated Omicron exposures override ancestral SARS-CoV-2 immune imprinting.

The continuing emergence of SARS-CoV-2 variants highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by vaccination based on the ancestral (hereafter referred to as WT) strain would compromise the antibody response to Omicron-based boosters1-5. Vaccination strategies to counter immune imprinting are critically needed. Here we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. In mice, the efficacy of single Omicron boosting is heavily limited when using variants that are antigenically distinct from WT-such as the XBB variant-and this concerning situation could be mitigated by a second Omicron booster. Similarly, in humans, repeated Omicron infections could alleviate WT vaccination-induced immune imprinting and generate broad neutralization responses in both plasma and nasal mucosa. Notably, deep mutational scanning-based epitope characterization of 781 receptor-binding domain (RBD)-targeting monoclonal antibodies isolated from repeated Omicron infection revealed that double Omicron exposure could induce a large proportion of matured Omicron-specific antibodies that have distinct RBD epitopes to WT-induced antibodies. Consequently, immune imprinting was largely mitigated, and the bias towards non-neutralizing epitopes observed in single Omicron exposures was restored. On the basis of the deep mutational scanning profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated that these mutations could further boost the immune-evasion capability of XBB.1.5 while maintaining high ACE2-binding affinity. Our findings suggest that the WT component should be abandoned when updating COVID-19 vaccines, and individuals without prior Omicron exposure should receive two updated vaccine boosters.

Antigenicity and receptor affinity of SARS-CoV-2 BA.2.86 spike.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant, BA.2.86, has emerged and spread to numerous countries worldwide, raising alarm because its spike protein contains 34 additional mutations compared with its BA.2 predecessor1. We examined its antigenicity using human sera and monoclonal antibodies (mAbs). Reassuringly, BA.2.86 was no more resistant to human sera than the currently dominant XBB.1.5 and EG.5.1, indicating that the new subvariant would not have a growth advantage in this regard. Importantly, sera from people who had XBB breakthrough infection exhibited robust neutralizing activity against all viruses tested, suggesting that upcoming XBB.1.5 monovalent vaccines could confer added protection. Although BA.2.86 showed greater resistance to mAbs to subdomain 1 (SD1) and receptor-binding domain (RBD) class 2 and 3 epitopes, it was more sensitive to mAbs to class 1 and 4/1 epitopes in the 'inner face' of the RBD that is exposed only when this domain is in the 'up' position. We also identified six new spike mutations that mediate antibody resistance, including E554K that threatens SD1 mAbs in clinical development. The BA.2.86 spike also had a remarkably high receptor affinity. The ultimate trajectory of this new SARS-CoV-2 variant will soon be revealed by continuing surveillance, but its worldwide spread is worrisome.

Broadly neutralizing antibody epitopes on HIV-1 particles are exposed after virus interaction with host cells.

The envelope (Env) glycoproteins on HIV-1 virions are the sole target of broadly neutralizing antibodies (bNAbs) and the focus of vaccines. However, many cross-reactive conserved epitopes are often occluded on virus particles, contributing to the evasion of humoral immunity. This study aimed to identify the Env epitopes that are exposed/occluded on HIV-1 particles and to investigate the mechanisms contributing to their masking. Using a flow cytometry-based assay, three HIV-1 isolates, and a panel of antibodies, we show that only select epitopes, including V2i, the gp120-g41 interface, and gp41-MPER, are accessible on HIV-1 particles, while V3, V2q, and select CD4bs epitopes are masked. These epitopes become accessible after allosteric conformational changes are induced by the pre-binding of select Abs, prompting us to test if similar conformational changes are required for these Abs to exhibit their neutralization capability. We tested HIV-1 neutralization where the virus-mAb mix was pre-incubated/not pre-incubated for 1 hour prior to adding the target cells. Similar levels of neutralization were observed under both assay conditions, suggesting that the interaction between virus and target cells sensitizes the virions for neutralization via bNAbs. We further show that lectin-glycan interactions can also expose these epitopes. However, this effect is dependent on the lectin specificity. Given that, bNAbs are ideal for providing sterilizing immunity and are the goal of current HIV-1 vaccine efforts, these data offer insight on how HIV-1 may occlude these vulnerable epitopes from the host immune response. In addition, the findings can guide the formulation of effective antibody combinations for therapeutic use. IMPORTANCE The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein mediates viral entry and is the sole target of neutralizing antibodies. Our data suggest that antibody epitopes including V2q (e.g., PG9, PGT145), CD4bs (e.g., VRC01, 3BNC117), and V3 (2219, 2557) are masked on HIV-1 particles. The PG9 and 2219 epitopes became accessible for binding after conformational unmasking was induced by the pre-binding of select mAbs. Attempts to understand the masking mechanism led to the revelation that interaction between virus and host cells is needed to sensitize the virions for neutralization by broadly neutralizing antibodies (bNAbs). These data provide insight on how bNAbs may gain access to these occluded epitopes to exert their neutralization effects and block HIV-1 infection. These findings have important implications for the way we evaluate the neutralizing efficacy of antibodies and can potentially guide vaccine design.

Vaccination with prefusion-stabilized respiratory syncytial virus fusion protein elicits antibodies targeting a membrane-proximal epitope.

IMPORTANCE: Respiratory syncytial virus (RSV) is the leading cause of bronchiolitis and pneumonia in infants, infecting all children by age 5. RSV also causes substantial morbidity and mortality in older adults, and a vaccine for older adults based on a prefusion-stabilized form of the viral F glycoprotein was recently approved by the FDA. Here, we investigate a set of antibodies that belong to the same public clonotype and were isolated from individuals vaccinated with a prefusion-stabilized RSV F protein. Our results reveal that these antibodies are highly potent and recognize a previously uncharacterized antigenic site on the prefusion F protein. Vaccination with prefusion RSV F proteins appears to boost the elicitation of these neutralizing antibodies, which are not commonly elicited by natural infection.

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.

Critical review of conformational B-cell epitope prediction methods.

Accurate in silico prediction of conformational B-cell epitopes would lead to major improvements in disease diagnostics, drug design and vaccine development. A variety of computational methods, mainly based on machine learning approaches, have been developed in the last decades to tackle this challenging problem. Here, we rigorously benchmarked nine state-of-the-art conformational B-cell epitope prediction webservers, including generic and antibody-specific methods, on a dataset of over 250 antibody-antigen structures. The results of our assessment and statistical analyses show that all the methods achieve very low performances, and some do not perform better than randomly generated patches of surface residues. In addition, we also found that commonly used consensus strategies that combine the results from multiple webservers are at best only marginally better than random. Finally, we applied all the predictors to the SARS-CoV-2 spike protein as an independent case study, and showed that they perform poorly in general, which largely recapitulates our benchmarking conclusions. We hope that these results will lead to greater caution when using these tools until the biases and issues that limit current methods have been addressed, promote the use of state-of-the-art evaluation methodologies in future publications and suggest new strategies to improve the performance of conformational B-cell epitope prediction methods.

Design of a multi-epitope Zika virus vaccine candidate - an in-silico study.

Zika virus (ZIKV), an RNA virus, rapidly spreads Aedes mosquito-borne sickness. Currently, there are neither effective vaccines nor therapeutics available to prevent or treat ZIKV infection. In this study, to address these unmet medical needs, we aimed to design B- and T-cell candidate multi-epitope-based subunit against ZIKV using an in silico approach. In this study we applied immunoinformatics, molecular docking, and dynamic simulation assessments targeting the most immunogenic proteins; the capsid (C), envelope (E) proteins and the non-stuctural protein (NS1), described in our previous study, and which predicted immunodominant B and T cell epitopes. The final non-allergenic and highly antigenic multi-epitope was constituted of immunogenic screened-epitopes (3 CTL and 3 HTL) and the ß-defensin as an adjuvant that have been linked using EAAAK, AAY, and GPGPG linkers, respectively. The final construct containing 143 amino acids was characterized for its allergenicity, antigenicity, and physiochemical properties; and found to be safe and immunogenic with a good prediction of solubility. The existence of IFN-γ epitopes asserts the capacity to trigger strong immune responses. Subsequently, the molecular docking among vaccine and immune receptors (TLR2/TLR4) was revealed with a good binding affinity with and stable molecular interactions. Molecular dynamics simulation confirmed the stability of the complexes. Finally, the construct was subjected to in silico cloning demonstrating the efficiently of its expression in E.coli. However, this study needs the experimental validation to demonstrate vaccine safety and efficacy.Communicated by Ramaswamy H. Sarma.

Antibody feedback regulates immune memory after SARS-CoV-2 mRNA vaccination.

Feedback inhibition of humoral immunity by antibodies was first documented in 19091. Subsequent studies showed that, depending on the context, antibodies can enhance or inhibit immune responses2,3. However, little is known about how pre-existing antibodies influence the development of memory B cells. Here we examined the memory B cell response in individuals who received two high-affinity anti-SARS-CoV-2 monoclonal antibodies and subsequently two doses of an mRNA vaccine4-8. We found that the recipients of the monoclonal antibodies produced antigen-binding and neutralizing titres that were only fractionally lower compared than in control individuals. However, the memory B cells of the individuals who received the monoclonal antibodies differed from those of control individuals in that they predominantly expressed low-affinity IgM antibodies that carried small numbers of somatic mutations and showed altered receptor binding domain (RBD) target specificity, consistent with epitope masking. Moreover, only 1 out of 77 anti-RBD memory antibodies tested neutralized the virus. The mechanism underlying these findings was examined in experiments in mice that showed that germinal centres formed in the presence of the same antibodies were dominated by low-affinity B cells. Our results indicate that pre-existing high-affinity antibodies bias germinal centre and memory B cell selection through two distinct mechanisms: (1) by lowering the activation threshold for B cells, thereby permitting abundant lower-affinity clones to participate in the immune response; and (2) through direct masking of their cognate epitopes. This may in part explain the shifting target profile of memory antibodies elicited by booster vaccinations9.

Preparation of monoclonal antibody and identification of two novel B cell epitopes to VP1 protein of porcine sapelovirus.

Porcine sapelovirus (PSV) is an important emerging swine pathogen that causes diarrhoea, respiratory distress, severe reproductive system and neurological disorders in pigs, posing huge threat to swine industry. However, there are no effective serological diagnostic products and the epitope characterization of PSV VP1 protein is still largely unknown. In current study, we successfully expressed recombinant His-VP1 protein by prokaryotic expression system and the recombinant VP1 protein had good immunogenicity. BALB/C mice were then selected and immunized with purified recombinant VP1 protein, and two monoclonal antibodies (Mabs) 9F10 and 15E4 against VP1 were successfully prepared by hybrioma technology. The isotype of these two Mabs were identified and showed that Mab 9F10 with the heavy chain subtype was IgG1 and the light chain subtype was kappa. Mab 15E4 was identified as IgG2 for the heavy chain subtype and Kappa for the light chain subtype. The antigen epitopes of prepared two VP1 Mabs were clearly identified. The minimal unit of B cell specific epitope recognized by Mab 15E4 was 203YDGDG207 and conserved in different strain genotypes of PSV, indicating this epitope may be a good target for serological detection of PSV. However, the epitope recognized by Mab 9F10 was 8QAIVNRT14 and varied greatly among different PSV strains. Structural modeling analysis showed that the identified two novel B cell epitopes were located on the surface of VP1. Our study provides useful tool for the establishment the serological detection methods of PSV and may support the study of VP1 protein function.

Long-primed germinal centres with enduring affinity maturation and clonal migration.

Germinal centres are the engines of antibody evolution. Here, using human immunodeficiency virus (HIV) Env protein immunogen priming in rhesus monkeys followed by a long period without further immunization, we demonstrate germinal centre B (BGC) cells that last for at least 6 months. A 186-fold increase in BGC cells was present by week 10 compared with conventional immunization. Single-cell transcriptional profiling showed that both light- and dark-zone germinal centre states were sustained. Antibody somatic hypermutation of BGC cells continued to accumulate throughout the 29-week priming period, with evidence of selective pressure. Env-binding BGC cells were still 49-fold above baseline at 29 weeks, which suggests that they could remain active for even longer periods of time. High titres of HIV-neutralizing antibodies were generated after a single booster immunization. Fully glycosylated HIV trimer protein is a complex antigen, posing considerable immunodominance challenges for B cells1,2. Memory B cells generated under these long priming conditions had higher levels of antibody somatic hypermutation, and both memory B cells and antibodies were more likely to recognize non-immunodominant epitopes. Numerous BGC cell lineage phylogenies spanning more than the 6-month germinal centre period were identified, demonstrating continuous germinal centre activity and selection for at least 191 days with no further antigen exposure. A long-prime, slow-delivery (12 days) immunization approach holds promise for difficult vaccine targets and suggests that patience can have great value for tuning of germinal centres to maximize antibody responses.

Rotavirus VP4 Epitope of a Broadly Neutralizing Human Antibody Defined by Its Structure Bound with an Attenuated-Strain Virion.

Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* ß-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the "upright" conformation present on fully infectious virions to the "reversed" conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.

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.

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.

Predicted 3D model of the M protein of Porcine Epidemic Diarrhea Virus and analysis of its immunogenic potential.

The membrane protein M of the Porcine Epidemic Diarrhea Virus (PEDV) is the most abundant component of the viral envelope. The M protein plays a central role in the morphogenesis and assembly of the virus through protein interactions of the M-M, M-Spike (S) and M-nucleocapsid (N) type. The M protein is known to induce protective antibodies in pigs and to participate in the antagonistic response of the cellular antiviral system coordinated by the type I and type III interferon pathways. The 3D structure of the PEDV M protein is still unknown. The present work exposes a predicted 3D model of the M protein generated using the Robetta protocol. The M protein model is organized into a transmembrane and a globular region. The obtained 3D model of the PEDV M protein was compared with 3D models of the SARS-CoV-2 M protein created using neural networks and with initial machine learning-based models created using trRosetta. The 3D model of the present study predicted four linear B-cell epitopes (RSVNASSGTG and KHGDYSAVSNPSALT peptides are noteworthy), six discontinuous B-cell epitopes, forty weak binding and fourteen strong binding T-cell epitopes in the CV777 M protein. A high degree of conservation of the epitopes predicted in the PEDV M protein was observed among different PEDV strains isolated in different countries. The data suggest that the M protein could be a potential candidate for the development of new treatments or strategies that activate protective cellular mechanisms against viral diseases.

Omicron escapes the majority of existing SARS-CoV-2 neutralizing antibodies.

The SARS-CoV-2 B.1.1.529 (Omicron) variant contains 15 mutations of the receptor-binding domain (RBD). How Omicron evades RBD-targeted neutralizing antibodies requires immediate investigation. Here we use high-throughput yeast display screening1,2 to determine the profiles of RBD escaping mutations for 247 human anti-RBD neutralizing antibodies and show that the neutralizing antibodies can be classified by unsupervised clustering into six epitope groups (A-F)-a grouping that is highly concordant with knowledge-based structural classifications3-5. Various single mutations of Omicron can impair neutralizing antibodies of different epitope groups. Specifically, neutralizing antibodies in groups A-D, the epitopes of which overlap with the ACE2-binding motif, are largely escaped by K417N, G446S, E484A and Q493R. Antibodies in group E (for example, S309)6 and group F (for example, CR3022)7, which often exhibit broad sarbecovirus neutralizing activity, are less affected by Omicron, but a subset of neutralizing antibodies are still escaped by G339D, N440K and S371L. Furthermore, Omicron pseudovirus neutralization showed that neutralizing antibodies that sustained single mutations could also be escaped, owing to multiple synergetic mutations on their epitopes. In total, over 85% of the tested neutralizing antibodies were escaped by Omicron. With regard to neutralizing-antibody-based drugs, the neutralization potency of LY-CoV016, LY-CoV555, REGN10933, REGN10987, AZD1061, AZD8895 and BRII-196 was greatly undermined by Omicron, whereas VIR-7831 and DXP-604 still functioned at a reduced efficacy. Together, our data suggest that infection with Omicron would result in considerable humoral immune evasion, and that neutralizing antibodies targeting the sarbecovirus conserved region will remain most effective. Our results inform the development of antibody-based drugs and vaccines against Omicron and future variants.

Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift.

The recently emerged SARS-CoV-2 Omicron variant encodes 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), thereby raising concerns about the effectiveness of available vaccines and antibody-based therapeutics. Here we show that the Omicron RBD binds to human ACE2 with enhanced affinity, relative to the Wuhan-Hu-1 RBD, and binds to mouse ACE2. Marked reductions in neutralizing activity were observed against Omicron compared to the ancestral pseudovirus in plasma from convalescent individuals and from individuals who had been vaccinated against SARS-CoV-2, but this loss was less pronounced after a third dose of vaccine. Most monoclonal antibodies that are directed against the receptor-binding motif lost in vitro neutralizing activity against Omicron, with only 3 out of 29 monoclonal antibodies retaining unaltered potency, including the ACE2-mimicking S2K146 antibody1. Furthermore, a fraction of broadly neutralizing sarbecovirus monoclonal antibodies neutralized Omicron through recognition of antigenic sites outside the receptor-binding motif, including sotrovimab2, S2X2593 and S2H974. The magnitude of Omicron-mediated immune evasion marks a major antigenic shift in SARS-CoV-2. Broadly neutralizing monoclonal antibodies that recognize RBD epitopes that are conserved among SARS-CoV-2 variants and other sarbecoviruses may prove key to controlling the ongoing pandemic and future zoonotic spillovers.

Scoping review of the applications of peptide microarrays on the fight against human infections.

INTRODUCTION: This scoping review explores the use of peptide microarrays in the fight against infectious diseases. The research domains explored included the use of peptide microarrays in the mapping of linear B-cell and T cell epitopes, antimicrobial peptide discovery, immunosignature characterisation and disease immunodiagnostics. This review also provides a short overview of peptide microarray synthesis. METHODS: Electronic databases were systematically searched to identify relevant studies. The review was conducted using the Joanna Briggs Institute methodology for scoping reviews and data charting was performed using a predefined form. The results were reported by narrative synthesis in line with the Preferred Reporting Items for Systematic reviews and Meta-Analyses extension for Scoping Reviews guidelines. RESULTS: Ninety-five articles from 103 studies were included in the final data charting process. The majority (92. 0%) of the articles were published during 2010-2020 and were mostly from Europe (44.2%) and North America (34.7%). The findings were from the investigation of viral (45.6%), bacterial (32. 0%), parasitic (23.3%) and fungal (2. 0%) infections. Out of the serological studies, IgG was the most reported antibody type followed by IgM. The largest portion of the studies (77.7%) were related to mapping B-cell linear epitopes, 5.8% were on diagnostics, 5.8% reported on immunosignature characterisation and 8.7% reported on viral and bacterial cell binding assays. Two studies reported on T-cell epitope profiling. CONCLUSION: The most important application of peptide microarrays was found to be B-cell epitope mapping or antibody profiling to identify diagnostic and vaccine targets. Immunosignatures identified by random peptide microarrays were found to be applied in the diagnosis of infections and interrogation of vaccine responses. The analysis of the interactions of random peptide microarrays with bacterial and viral cells using binding assays enabled the identification of antimicrobial peptides. Peptide microarray arrays were also used for T-cell linear epitope mapping which may provide more information for the design of peptide-based vaccines and for the development of diagnostic reagents.

In silico construction of a multiepitope Zika virus vaccine using immunoinformatics tools.

Zika virus (ZIKV) is an arbovirus from the Flaviviridae family and Flavivirus genus. Neurological events have been associated with ZIKV-infected individuals, such as Guillain-Barré syndrome, an autoimmune acute neuropathy that causes nerve demyelination and can induce paralysis. With the increase of ZIKV infection incidence in 2015, malformation and microcephaly cases in newborns have grown considerably, which suggested congenital transmission. Therefore, the development of an effective vaccine against ZIKV became an urgent need. Live attenuated vaccines present some theoretical risks for administration in pregnant women. Thus, we developed an in silico multiepitope vaccine against ZIKV. All structural and non-structural proteins were investigated using immunoinformatics tools designed for the prediction of CD4 + and CD8 + T cell epitopes. We selected 13 CD8 + and 12 CD4 + T cell epitopes considering parameters such as binding affinity to HLA class I and II molecules, promiscuity based on the number of different HLA alleles that bind to the epitopes, and immunogenicity. ZIKV Envelope protein domain III (EDIII) was added to the vaccine construct, creating a hybrid protein domain-multiepitope vaccine. Three high scoring continuous and two discontinuous B cell epitopes were found in EDIII. Aiming to increase the candidate vaccine antigenicity even further, we tested secondary and tertiary structures and physicochemical parameters of the vaccine conjugated to four different protein adjuvants: flagellin, 50S ribosomal protein L7/L12, heparin-binding hemagglutinin, or RS09 synthetic peptide. The addition of the flagellin adjuvant increased the vaccine's predicted antigenicity. In silico predictions revealed that the protein is a probable antigen, non-allergenic and predicted to be stable. The vaccine's average population coverage is estimated to be 87.86%, which indicates it can be administered worldwide. Peripheral Blood Mononuclear Cells (PBMC) of individuals with previous ZIKV infection were tested for cytokine production in response to the pool of CD4 and CD8 ZIKV peptide selected. CD4 + and CD8 + T cells showed significant production of IFN-γ upon stimulation and IL-2 production was also detected by CD8 + T cells, which indicated the potential of our peptides to be recognized by specific T cells and induce immune response. In conclusion, we developed an in silico universal vaccine predicted to induce broad and high-coverage cellular and humoral immune responses against ZIKV, which can be a good candidate for posterior in vivo validation.

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.

Immunoinformatics based designing a multi-epitope vaccine against pathogenic Chandipura vesiculovirus.

Chandipura vesiculovirus (CHPV) is a rapidly emerging pathogen responsible for causing acute encephalitis. Due to its widespread occurrence in Asian and African countries, this has become a global threat, and there is an urgent need to design an effective and nonallergenic vaccine against this pathogen. The present study aimed to develop a multi-epitope vaccine using an immunoinformatics approach. The conventional method of vaccine design involves large proteins or whole organism which leads to unnecessary antigenic load with increased chances of allergenic reactions. In addition, the process is also very time-consuming and labor-intensive. These limitations can be overcome by peptide-based vaccines comprising short immunogenic peptide fragments that can elicit highly targeted immune responses, avoiding the chances of allergenic reactions, in a relatively shorter time span. The multi-epitope vaccine constructed using CTL, HTL, and IFN-γ epitopes was able to elicit specific immune responses when exposed to the pathogen, in silico. Not only that, molecular docking and molecular dynamics simulation studies confirmed a stable interaction of the vaccine with the immune receptors. Several physicochemical analyses of the designed vaccine candidate confirmed it to be highly immunogenic and nonallergic. The computer-aided analysis performed in this study suggests that the designed multi-epitope vaccine can elicit specific immune responses and can be a potential candidate against CHPV.