Epitope Variants Research Articles

Structural assessment of HLA-A2-restricted SARS-CoV-2 spike epitopes recognized by public and private T-cell receptors

T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide–MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.

Mechanistic insights into the digestion of complex dietary fibre by the rumen microbiota using combinatorial high-resolution glycomics and transcriptomic analyses

There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan.This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

Complete genomic analysis of uncommon G12P[11] rotavirus causing a nosocomial outbreak of acute diarrhea in the newborns in New Delhi, India.

Rotaviruses (RVs) are the major causative agents of acute gastroenteritis in children, but in neonates, RV infections are generally nosocomial in origin and mostly asymptomatic. However, there have been infrequent reports of nosocomial outbreaks of clinical disease in this population. In this study, we describe uncommon RV genotype; G12P[11] associated with an outbreak of acute gastroenteritis in the neonatal ward and neonatal intensive care unit (NICU) in New Delhi, North India. Full-genome analyses of the pathogenic G12P[11] strain was carried out to map the genotype constellation and further to explore the variations in the antigenic epitopes on the immunodominant VP7 and VP4 proteins, the amino acid sequences were compared with neonatal strains; ROTAVAC® (G9P[11]) and asymptomatic G12P[11] and also other G/P-type matched strains. The study revealed G12-P[11]-I1-R1-C1-M1-A1-N1-T1-E1-H1 human Wa-like genotype constellation and highlights evidence of gene reassortment. No significant differences were observed in the sequences of structural (except VP3) and nonstructural encoding genes of G12P[11] strains recovered from symptomatic and asymptomatic neonates. Presence of additional N-linked glycosylation site was noted in the G12 strains, as a consequence of a change from Asp→Asn at amino acid position 238. Interestingly, only two and four amino acids substitution within the 7-1a and 8-1 antigenic epitope were observed, respectively, compared with asymptomatic G12P[11] strain. The study emphasizes the importance of close monitoring of RV outbreaks in neonates for early alarming of novel strain.

High-Throughput Surface Plasmon Resonance Biosensors for Identifying Diverse Therapeutic Monoclonal Antibodies.

Identification of antibodies targeting diverse functional epitopes on an antigen is highly crucial for discovering effective therapeutic candidates. Employing a traditional stepwise antibody "screening funnel" as well as prioritizing affinity-based selections over epitope-based selections, result in lead antibody panels lacking epitope diversity. In the present study, we employed an array-based surface plasmon resonance (SPR) platform to perform high-throughput epitope binning analysis on a large number of monoclonal antibodies (mAbs) generated in the early drug discovery process. The mAb panel contained clones from different antibody generation techniques and diverse transgenic mouse strains. The epitope binning results were analyzed in unique ways using various visualizations in the form of dendrograms and network plots, which assisted in determining diversity and redundancy in the mAb sample set. The binning data were further integrated with affinity information to evaluate the performance of seven different transgenic mouse strains. The combination of epitope binning results with binding kinetics and sequence analysis provided an effective and efficient way of selecting high affinity antibodies representing a diverse set of sequence families and epitopes.

Immunogenicity and Protective Potential of Mucosal Vaccine Formulations Based on Conserved Epitopes of Influenza A Viruses Fused to an Innovative Ring Nanoplatform in Mice and Chickens.

Current inactivated vaccines against influenza A viruses (IAV) mainly induce immune responses against highly variable epitopes across strains and are mostly delivered parenterally, limiting the development of an effective mucosal immunity. In this study, we evaluated the potential of intranasal formulations incorporating conserved IAV epitopes, namely the long alpha helix (LAH) of the stalk domain of hemagglutinin and three tandem repeats of the ectodomain of the matrix protein 2 (3M2e), as universal mucosal anti-IAV vaccines in mice and chickens. The IAV epitopes were grafted to nanorings, a novel platform technology for mucosal vaccination formed by the nucleoprotein (N) of the respiratory syncytial virus, in fusion or not with the C-terminal end of the P97 protein (P97c), a recently identified Toll-like receptor 5 agonist. Fusion of LAH to nanorings boosted the generation of LAH-specific systemic and local antibody responses as well as cellular immunity in mice, whereas the carrier effect of nanorings was less pronounced towards 3M2e. Mice vaccinated with chimeric nanorings bearing IAV epitopes in fusion with P97c presented modest LAH- or M2e-specific IgG titers in serum and were unable to generate a mucosal humoral response. In contrast, N-3M2e or N-LAH nanorings admixed with Montanide™ gel (MG) triggered strong specific humoral responses, composed of serum type 1/type 2 IgG and mucosal IgG and IgA, as well as cellular responses dominated by type 1/type 17 cytokine profiles. All mice vaccinated with the [N-3M2e + N-LAH + MG] formulation survived an H1N1 challenge and the combination of both N-3M2e and N-LAH nanorings with MG enhanced the clinical and/or virological protective potential of the preparation in comparison to individual nanorings. Chickens vaccinated parenterally or mucosally with N-LAH and N-3M2e nanorings admixed with Montanide™ adjuvants developed a specific systemic humoral response, which nonetheless failed to confer protection against heterosubtypic challenge with a highly pathogenic H5N8 strain. Thus, while the combination of N-LAH and N-3M2e nanorings with Montanide™ adjuvants shows promise as a universal mucosal anti-IAV vaccine in the mouse model, further experiments have to be conducted to extend its efficacy to poultry.

The impact of viral mutations on recognition by SARS-CoV-2 specific T cells

SummaryWe identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.

Https://onco-hema.healthbooktimes.org/article/29047-mrna-vaccines-against-infectious-diseases-and-cancer?auth_token=GXnDwFXw-1WxSg9ozO7O

Synthetic mRNA acts as a template for synthesizing proteins, protein fragments, or peptides and now has many pharmaceutical applications.^1,2^ Coronavirus disease 2019 (COVID-19) due to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel zoonotic RNA virus, has resulted in the rapid development of dedicated mRNA vaccines.^3–5^ This rapid response was made possible by using mRNA platforms that already existed for experimental vaccines against other infectious diseases and cancer.^6–12^ Carrier-based mRNA vaccines have been developed using lipid-based delivery, peptide-based delivery, polymer-based delivery, and cationic nano-emulsions, as well as dendritic cells.^13^ The mRNA vaccine leads to the expression of encoded antigens in antigen-presenting cells (APCs), generating both innate and adaptive immune responses. Future developments in mRNA therapy in oncology are expected to include adaptations in the routes of administration and co-delivery of multiple mRNAs with other anti-cancer treatments, such as immune checkpoint inhibitors (ICI), radiotherapy, or chemotherapy. In addition, advances in next-generation sequencing (NGS) technology allow the genome, exome, and transcriptome of a single cancer patient to be deciphered. This new knowledge about the diversity of epitopes in different tumors and corresponding specific T cells has allowed the advancement of personalized cancer treatments.^14^ The article aims to present the rationale for the new therapeutic roles of mRNA vaccines, from COVID-19 and other infections to personalized oncology therapeutics.

Antibody-dependent cellular cytotoxicity response to SARS-CoV-2 in COVID-19 patients

Antibody-dependent cellular cytotoxicity (ADCC) responses to viral infection are a form of antibody regulated immune responses mediated through the Fc fragment. Whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) triggered ADCC responses contributes to COVID-19 disease development is currently not well understood. To understand the potential correlation between ADCC responses and COVID-19 disease development, we analyzed the ADCC activity and neutralizing antibody response in 255 individuals ranging from asymptomatic to fatal infections over 1 year post disease. ADCC was elicited by 10 days post-infection, peaked by 11–20 days, and remained detectable until 400 days post-infection. In general, patients with severe disease had higher ADCC activities. Notably, patients who had severe disease and recovered had higher ADCC activities than patients who had severe disease and deceased. Importantly, ADCC activities were mediated by a diversity of epitopes in SARS-COV-2-infected mice and induced to comparable levels against SARS-CoV-2 variants of concern (VOCs) (B.1.1.7, B.1.351, and P.1) as that against the D614G mutant in human patients and vaccinated mice. Our study indicates anti-SARS-CoV-2 ADCC as a major trait of COVID-19 patients with various conditions, which can be applied to estimate the extra-neutralization level against COVID-19, especially lethal COVID-19.

Broad cross-reactivity across sarbecoviruses exhibited by a subset of COVID-19 donor-derived neutralizing antibodies.

Many anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) neutralizing antibodies target the angiotensin-converting enzyme 2 (ACE2) binding site on viral spike receptor-binding domains (RBDs). Potent antibodies recognize exposed variable epitopes, often rendering them ineffective against other sarbecoviruses and SARS-CoV-2 variants. Class 4 anti-RBD antibodies against a less-exposed, but more-conserved, cryptic epitope could recognize newly emergent zoonotic sarbecoviruses and variants, but they usually show only weak neutralization potencies. Here, we characterize two class 4 anti-RBD antibodies derived from coronavirus disease 2019 (COVID-19) donors that exhibit breadth and potent neutralization of zoonotic coronaviruses and SARS-CoV-2 variants. C118-RBD and C022-RBD structures reveal orientations that extend from the cryptic epitope to occlude ACE2 binding and CDRH3-RBD main-chain H-bond interactions that extend an RBD β sheet, thus reducing sensitivity to RBD side-chain changes. A C118-spike trimer structure reveals rotated RBDs that allow access to the cryptic epitope and the potential for intra-spike crosslinking to increase avidity. These studies facilitate vaccine design and illustrate potential advantages of class 4 RBD-binding antibody therapeutics.

Antibody responses induced by SHIV infection are more focused than those induced by soluble native HIV-1 envelope trimers in non-human primates.

The development of an effective human immunodeficiency virus (HIV-1) vaccine is a high global health priority. Soluble native-like HIV-1 envelope glycoprotein trimers (Env), including those based on the SOSIP design, have shown promise as vaccine candidates by inducing neutralizing antibody responses against the autologous virus in animal models. However, to overcome HIV-1's extreme diversity a vaccine needs to induce broadly neutralizing antibodies (bNAbs). Such bNAbs can protect non-human primates (NHPs) and humans from infection. The prototypic BG505 SOSIP.664 immunogen is based on the BG505 env sequence isolated from an HIV-1-infected infant from Kenya who developed a bNAb response. Studying bNAb development during natural HIV-1 infection can inform vaccine design, however, it is unclear to what extent vaccine-induced antibody responses to Env are comparable to those induced by natural infection. Here, we compared Env antibody responses in BG505 SOSIP-immunized NHPs with those in BG505 SHIV-infected NHPs, by analyzing monoclonal antibodies (mAbs). We observed three major differences between BG505 SOSIP immunization and BG505 SHIV infection. First, SHIV infection resulted in more clonal expansion and less antibody diversity compared to SOSIP immunization, likely because of higher and/or prolonged antigenic stimulation and increased antigen diversity during infection. Second, while we retrieved comparatively fewer neutralizing mAbs (NAbs) from SOSIP-immunized animals, these NAbs targeted more diverse epitopes compared to NAbs from SHIV-infected animals. However, none of the NAbs, either elicited by vaccination or infection, showed any breadth. Finally, SOSIP immunization elicited antibodies against the base of the trimer, while infection did not, consistent with the base being placed onto the virus membrane in the latter setting. Together these data provide new insights into the antibody response against BG505 Env during infection and immunization and limitations that need to be overcome to induce better responses after vaccination.

Generation of cancer vaccine immunogens derived from Oncofetal antigen (OFA/iLRP) using variable epitope libraries tested in an aggressive breast cancer model

After decades of cancer vaccine efforts, there is an imperious necessity for novel ideas that may result in better tumor control in patients. We have proposed the use of a novel Variable Epitope Library (VEL) vaccine strategy, which incorporates an unprecedented number of mutated epitopes to target antigenic variability and break tolerance against tumor-associated antigens. Here, we used an oncofetal antigen/immature laminin receptor protein-derived sequence to generate 9-mer and 43-mer VEL immunogens. 4T1 tumor-bearing mice developed epitope-specific CD8+IFN-γ+ and CD4+IFN-γ+ T cell responses after treatment. Tumor and lung analysis demonstrated that VELs could increase the number of tumor-infiltrating lymphocytes with diverse effector functions while reducing the number of immunosuppressive myeloid-derived suppressor and regulatory T cells. Most importantly, VEL immunogens inhibited tumor growth and metastasis after a single dose. The results presented here are consistent with our previous studies and provide evidence for VEL immunogens’ feasibility as promising cancer immunotherapy.

Mapping Epitopes Recognised by Autoantibodies Shows Potential for the Diagnosis of High-Grade Serous Ovarian Cancer and Monitoring Response to Therapy for This Malignancy.

Simple SummaryMost women are diagnosed with high-grade serous ovarian cancer (HGSOC) at stage III, when the cancer has already spread, contributing to poor survival outcomes. However, while earlier diagnosis increases survival rates, there is a lack of early diagnosis biomarkers. Previously, autoantibodies specific for phosphorylated heat shock factor 1 (HSF1-PO4) were suggested as a potential diagnostic biomarker for early-stage HGSOC. In the present study, specific regions within HSF1 were identified, tested and confirmed as useful biomarkers, with comparable diagnostic potential to the full protein, across two separate clinical cohorts. Additionally, antibody responses to HSF1-PO4 and the corresponding peptides were found to increase following a round of standard first-line chemotherapy. Together, our data suggest that the identified short peptide sequences could be used as practical alternatives to support early diagnosis or monitor responses to chemotherapy.Autoantibodies recognising phosphorylated heat shock factor 1 (HSF1-PO4) protein are suggested as potential new diagnostic biomarkers for early-stage high-grade serous ovarian cancer (HGSOC). We predicted in silico B-cell epitopes in human and murine HSF1. Three epitope regions were synthesised as peptides. Circulating immunoglobulin A (cIgA) against the predicted peptide epitopes or HSF1-PO4 was measured using ELISA, across two small human clinical trials of HGSOC patients at diagnosis. To determine whether chemotherapy would promote changes in reactivity to either HSF1-PO4 or the HSF-1 peptide epitopes, IgA responses were further assessed in a sample of patients after a full cycle of chemotherapy. Anti-HSF1-PO4 responses correlated with antibody responses to the three selected epitope regions, regardless of phosphorylation, with substantial cross-recognition of the corresponding human and murine peptide epitope variants. Assessing reactivity to individual peptide epitopes, compared to HSF1-PO4, improved assay sensitivity. IgA responses to HSF1-PO4 further increased significantly post treatment, indicating that HSF1-PO4 is a target for immunity in response to chemotherapy. Although performed in a small cohort, these results offer potential insights into the interplay between autoimmunity and ovarian cancer and offer new peptide biomarkers for early-stage HGSOC diagnosis, to monitor responses to chemotherapy, and widely for pre-clinical HGSOC research.

Epitope diversity of SARS-CoV-2 hyperimmune intravenous human immunoglobulins and neutralization of variants of concern

SummaryHyperimmune immunoglobulin (hCoV-2IG) generated from SARS-CoV-2 convalescent plasma (CP) are under evaluation in clinical trials. Here we explored the antibody epitope repertoire, and virus neutralizing capacity of six hCoV-2IG batches as well as nine CP against SARS-CoV-2 and emerging variants of concern (VOCs). Epitope-mapping by gene-fragment phage display library spanning the SARS-CoV-2 spike demonstrated broad recognition of multiple antigenic sites spanning the entire spike that was higher for hCoV-2IG than CP, with predominant binding to the fusion peptide. In the pseudovirus neutralization assay and in the wild-type SARS-CoV-2 PRNT assay, hCoV-2IG lots showed higher titers against the WA-1 strain compared with CP. Neutralization of VOCs were reduced to different extent by hCoV-2IG lots but were higher than CP. Significant reduction of hCoV-2IG binding was observed to RBD-E484K followed by RBD-N501Y (but not RBD-K417N). This study suggests that post-exposure treatment with hCoV-2IG could be preferable to CP.

Understanding Antibody Responses in Early Life: Baby Steps towards Developing an Effective Influenza Vaccine

The immune system of young infants is both quantitatively and qualitatively distinct from that of adults, with diminished responsiveness leaving these individuals vulnerable to infection. Because of this, young infants suffer increased morbidity and mortality from respiratory pathogens such as influenza viruses. The impaired generation of robust and persistent antibody responses in these individuals makes overcoming this increased vulnerability through vaccination challenging. Because of this, an effective vaccine against influenza viruses in infants under 6 months is not available. Furthermore, vaccination against influenza viruses is challenging even in adults due to the high antigenic variability across viral strains, allowing immune evasion even after induction of robust immune responses. This has led to substantial interest in understanding how specific antibody responses are formed to variable and conserved components of influenza viruses, as immune responses tend to strongly favor recognition of variable epitopes. Elicitation of broadly protective antibody in young infants, therefore, requires that both the unique characteristics of young infant immunity as well as the antibody immunodominance present among epitopes be effectively addressed. Here, we review our current understanding of the antibody response in newborns and young infants and discuss recent developments in vaccination strategies that can modulate both magnitude and epitope specificity of IAV-specific antibody.

Mapping mutations to the SARS-CoV-2 RBD that escape binding by different classes of antibodies

Monoclonal antibodies targeting a variety of epitopes have been isolated from individuals previously infected with SARS-CoV-2, but the relative contributions of these different antibody classes to the polyclonal response remains unclear. Here we use a yeast-display system to map all mutations to the viral spike receptor-binding domain (RBD) that escape binding by representatives of three potently neutralizing classes of anti-RBD antibodies with high-resolution structures. We compare the antibody-escape maps to similar maps for convalescent polyclonal plasmas, including plasmas from individuals from whom some of the antibodies were isolated. While the binding of polyclonal plasma antibodies are affected by mutations across multiple RBD epitopes, the plasma-escape maps most resemble those of a single class of antibodies that target an epitope on the RBD that includes site E484. Therefore, although the human immune system can produce antibodies that target diverse RBD epitopes, in practice the polyclonal response to infection is skewed towards a single class of antibodies targeting an epitope that is already undergoing rapid evolution.

Dynamics of the Magnitude, Breadth and Depth of the Antibody Response at Epitope Level Following Dengue Infection.

Dengue is a major public health problem in tropical and sub-tropical regions worldwide. Since the Zika epidemic and the increased co-circulation of other arboviruses, the serology-based diagnosis of dengue has become more problematic due to the high antigenic resemblance, especially among the flavivirus family. Therefore, a more comprehensive understanding of the diversity, specificity and temporal evolution of the antibody response following dengue infection is needed. In order to close this knowledge gap, we used a high-density peptide microarray of 9,072 linear peptides covering the entire proteome diversity of dengue, Zika, yellow fever and chikungunya viruses. The IgM and IgG antibody responses were measured against the designed microarray in symptomatic dengue infected individuals from an arbovirus endemic area in Peru and in overseas travelers returning to Belgium, as representatives of multiple-exposed and primary infections, respectively. Serum samples were collected longitudinally across four time points over the period of six months in Peru and over two time points in travelers. We show that epitopes eliciting the strongest flavivirus cross-reactive antibodies, in both primary and secondary infections were concentrated in the capsid, E, NS1, NS3 and NS5 proteins. The IgG antibody responses against NS1 and NS3 followed a rise-and-fall pattern, with peak titers between two to four weeks after onset of illness. The response to the E and NS5 proteins increased rapidly in the acute phase and was maintained at stable levels until at least 6 months after illness. A more scattered IgM antibody reactivity across the viral proteome was observed in the acute phase of the disease and that persisted through the 6-month window. The magnitude, breadth (i.e. number of unique epitopes targeted) and depth (i.e. number of epitope variants recognized) of the IgG response was higher in secondary infections compared to primary infections. For IgM antibodies, the magnitude of the response was higher in primary infected individuals whereas the breadth and depth of the response was lower in this group compared with the endemic subjects. Finally, through this arboviral proteome-wide epitope mapping, we were able to identify IgM and IgG dengue-specific epitopes which can be useful serological markers for dengue diagnosis and serostatus determination.

COVID-19 as a Trigger of Brain Autoimmunity.

Entry of SARS-CoV-2 into the central nervous system (CNS) activates microglia, triggering chronic neuroinflammation and possibly neurodegeneration. The complex transcriptome of SARS-CoV-2 shares molecular similarities with diverse human CNS protein epitopes, leading to a cytokine storm and various autoantibodies, potentially culminating in an autoimmune state. A COVID-19 initiated CNS autoimmune cascade may occur via multiple pathways including molecular mimicry, bystander activation, epitope spreading, production of autoantibodies, and immortalization of effector B-cells.

Influence of DM-sensitivity on immunogenicity of MHC class II restricted antigens

BackgroundGraft-versus-host-disease (GvHD) is a major problem in allogeneic stem cell transplantation. We previously described two types of endogenous human leukocyte antigen (HLA)-II restricted antigens depending on their behavior towards HLA-DM....

Bispecific Antibodies with Native Human IgG Structure As Mimetics for FVIII Activity

Background: Non-factor therapies for hemophilia A are being evaluated to overcome the need for frequent i.v. administration and address therapeutic issues caused by anti-FVIII inhibitors. One possibility to substitute the lack of FVIII activity is the development of a bispecific antibody (biAb) that targets the two coagulation factors FIXa and FX. The two arms' binding characteristics and relative orientation are thought to be important to optimally bridge FIXa and FX and to at least partially mimic the function of FVIIIa, and facilitate FXa generation.Aim: To identify biAbs targeting FIXa and FX that have FVIII-mimetic activity using the κλ body platform, generating biAbs that are indistinguishable from native human IgG. As hemophilia A patients require life-long treatment, a native human IgG structure represents an advantage for the chronic treatment of this patient population.Methods: We used the κλ body platform to identify novel biAbs with FVIII-mimetic activity. First, antibodies against different forms of FIX(a) and FX(a) were selected using phage display. Then, κλ bodies were produced by co-expressing a common heavy chain and two light chains. As the κλ body is the major form secreted in the supernatant of the transfected cells, conveniently, cell culture supernatants were used directly for functional screening and multiple arms optimized in parallel.Results: Over 1000 unique antibodies were identified that target different epitopes on each factor, allowing for the generation of thousands of different biAbs combinations. To date, over 10,000 biAbs have been functionally screened. Unlike previous studies which only rarely identified FVIII-mimetic biAbs (Sampei et al., PLoS One 2013), the present approach resulted in isolation of ~350 unique arm combinations showing considerable FVIII mimetic activity. These results suggest that multiple geometries of the FIXa/FX biAb complex can lead to FVIII-mimetic activity.Conclusion: Theκλ body technology is optimal for generating native human and non-engineered biAbs of high epitope diversity, and exploring a wide range of geometries which is crucial to obtain biAbs for efficient FX activation. Efficient expression supernatant screening allowed multiple arms to be optimized in parallel and significantly improved FVIII-mimetic activity. The native human IgG structure of the FVIII-mimetic κλ bodies make them ideal candidates for long-term treatment of hemophilia A. DisclosuresKnappe:Shire: Employment. Fischer:GLG (Gerson Lehrman Group): Consultancy; Novimmune: Employment, Equity Ownership, Patents & Royalties, Research Funding. Douillard:Shire: Employment. Hartmann:Shire: Employment. Schrenk:Shire: Employment. Ravn:NovImmune: Employment, Equity Ownership. Magistrelli:Pierre Fabre: Patents & Royalties; NovImmune SA: Employment, Equity Ownership, Patents & Royalties. Gueneau:Novimmune: Employment, Equity Ownership. Didelot:Novimmune: Employment. Masternak:Novimmune: Employment, Equity Ownership. Kosco-Vilbois:Novimmune SA: Employment, Equity Ownership. Scheiflinger:Shire: Employment, Equity Ownership; Baxter: Equity Ownership. Dockal:Shire: Employment, Equity Ownership; Baxter: Equity Ownership, Patents & Royalties; Baxalta: Patents & Royalties.

Immune Evasion of SARS-CoV-2 Emerging Variants: What Have We Learnt So Far?

Despite the slow evolutionary rate of SARS-CoV-2 relative to other RNA viruses, its massive and rapid transmission during the COVID-19 pandemic has enabled it to acquire significant genetic diversity since it first entered the human population. This led to the emergence of numerous variants, some of them recently being labeled “variants of concern” (VOC), due to their potential impact on transmission, morbidity/mortality, and the evasion of neutralization by antibodies elicited by infection, vaccination, or therapeutic application. The potential to evade neutralization is the result of diversity of the target epitopes generated by the accumulation of mutations in the spike protein. While three globally recognized VOCs (Alpha or B.1.1.7, Beta or B.1.351, and Gamma or P.1) remain sensitive to neutralization albeit at reduced levels by the sera of convalescent individuals and recipients of several anti-COVID19 vaccines, the effect of spike variability is much more evident on the neutralization capacity of monoclonal antibodies. The newly recognized VOC Delta or lineage B.1.617.2, as well as locally accepted VOCs (Epsilon or B.1.427/29-US and B1.1.7 with the E484K-UK) are indicating the necessity of close monitoring of new variants on a global level. The VOCs characteristics, their mutational patterns, and the role mutations play in immune evasion are summarized in this review.