Receptor-binding Domain Research Articles

Genetic engineering employing MPB70 and its promoter enables efficient secretion and expression of foreign antigen in bacillus Calmette Guérin (BCG) Tokyo.

Vaccination is an important factor in public health. The recombinant bacillus Calmette Guérin (rBCG) vaccine, which expresses foreign antigens, is expected to be a superior vaccine against infectious diseases. Here, we report a new recombination platform in which the BCG Tokyo strain is transformed with nucleotide sequences encoding foreign protein fused with the MPB70 immunogenic protein precursor. By RNA-sequencing, mpb70 was found to be the most transcribed among all known genes of BCG Tokyo. Small oligopeptide, namely, polyhistidine tag, was able to be expressed in and secreted from rBCG through a process in which polyhistidine tag fused with intact MPB70 were transcribed by an mpb70 promoter. This methodology was applied to develop an rBCG expressing the receptor binding domain (RBD) of severe acute respiratory syndrome coronavirus 2. Immunoblotting images and mass spectrometry data showed that RBD was also secreted from rBCG. Sera from mice vaccinated with the rBCG showed a tendency of weak neutralizing capacity. The secretion was retained even after a freeze-drying process. The freeze-dried rBCG was administered to and recovered from mice. Recovered rBCG kept secreting RBD. Collectively, our recombination platform offers stable secretion of foreign antigens and can be applied to the development of practical rBCGs.

Analysis of related factors of plasma antibody levels in patients with severe and critical COVID-19

Coronavirus disease 2019 (COVID-19) continues to impact global public health. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become less virulent as it mutates, prompting China to ease restrictions at the end of 2022. With the complete reopening, a surge in COVID-19 cases has ensued. Therefore, we conducted a study to explore the correlation between plasma antibody levels and baseline conditions or clinical outcomes in severe and critical patients. We collected the basic information of 79 included patients. Enzyme-linked immunosorbent assay (ELISA) tests were performed on plasma samples. The receptor-binding domain (RBD) IgG antibody level of the mild group was significantly higher than that of the severe/critical group (P = 0.00049). And in the severe/critical group, there existed an association between plasma antibody levels and age (P < 0.001, r = − 0.471), as well as plasma antibody levels and vaccination status (P = 0.00147, eta2 = 0.211). Besides, the level of plasma antibody seemed to be moderately correlated with the age, indicating the need for heightened attention to infections in the elderly. And plasma antibody levels were strongly associated with vaccination status in the severe/critical patients.

SARS-CoV-2 receptor a distinct genetic profile specific to the Iraqi Kurdish population.

SARS-CoV-2, the virus responsible for COVID-19, enters host cells by binding its spike protein's receptor-binding domain (RBD) to the human angiotensin-converting enzyme 2 (ACE2) receptor's peptidase domain (PD). This interaction plays a crucial role in the virus's ability to invade host cells and establish infection. Numerous studies have identified specific residues crucial for their binding interaction. Our objective was to determine whether natural variations in the ACE2 receptor could impact its affinity for the S-protein RBD. To explore this, we focused on investigating the effects of natural variations in the ACE2 PD residues on its binding affinity to the S-protein RBD interface of SARS-CoV-2. We conducted a genotyping study in the Iraqi Kurdish population and identified significant genetic variations in key binding residues of the ACE2 PD residues, including N330K, K353R, R357Q, P389H, and R393H. These variations suggest a distinct genetic profile specific to the Kurdish population regarding their interaction with the SARS-CoV-2 virus. Understanding the implications of these variations is essential for comprehending the mechanisms of viral infection, developing targeted therapeutics, and refining treatment strategies and vaccine design. Additionally, studying these variations can provide insights into population-specific vulnerabilities, help monitor viral evolution and transmission, and guide the development of effective interventions.

Difference in the Dynamics of Antibody Titers between COVID-19 Vaccination and SARS-CoV-2 Infection in Healthcare Workers - A Case Study Based on Long-Term and Densely Repeated Measurements of Antibody Titers.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is prevalent worldwide, and effective and safe vaccines against this virus have been developed. Although trends in antibody titers after vaccination and/or SARS-CoV-2 infection have been reported, long-term studies with high frequency of measurements are limited. This report describes the long-term and detailed trends in the antibodies against SARS-CoV-2 S protein receptor-binding domain (S-RBD) measured repeatedly after vaccination and/or infection in 3 healthcare workers. All healthcare workers were administered 30 µg of the messenger RNA vaccine, BNT162b2, during all vaccinations. The peak value of the SARS-CoV-2 S-RBD titer was reached at 1-2 weeks after vaccination and then decreased by half within 8 weeks after vaccination; the peak values of the antibody titer increased with repeated vaccinations. In contrast, after SARS-CoV-2 infection, the peak value of the antibody titer was reached at 4-8 weeks after infection, and the antibody titer remained elevated up to 16-40 weeks after the peak. This report describes the long-term and detailed trends in the anti-SARS-CoV-2 S-RBD titers, showing different patterns after vaccination and/or SARS-CoV-2 infection.

Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds

Protein-based virus-like particles (P-VLPs) are commonly used to spatially organize antigens and enhance humoral immunity through multivalent antigen display. However, P-VLPs are thymus-dependent antigens that are themselves immunogenic and can induce B cell responses that may neutralize the platform. Here, we investigate thymus-independent DNA origami as an alternative material for multivalent antigen display using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, the primary target of neutralizing antibody responses. Sequential immunization of mice with DNA-based VLPs (DNA-VLPs) elicits protective neutralizing antibodies to SARS-CoV-2 in a manner that depends on the valency of the antigen displayed and on T cell help. Importantly, the immune sera do not contain boosted, class-switched antibodies against the DNA scaffold, in contrast to P-VLPs that elicit strong B cell memory against both the target antigen and the scaffold. Thus, DNA-VLPs enhance target antigen immunogenicity without generating scaffold-directed immunity and thereby offer an important alternative material for particulate vaccine design.

SARS-CoV-2 Spike Protein Stimulates Macropinocytosis in Murine and Human Macrophages via PKC-NADPH Oxidase Signaling.

Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While recent studies have demonstrated that SARS-CoV-2 may enter kidney and colon epithelial cells by inducing receptor-independent macropinocytosis, it remains unknown whether this process also occurs in cell types directly relevant to SARS-CoV-2-associated lung pneumonia, such as alveolar epithelial cells and macrophages. The goal of our study was to investigate the ability of SARS-CoV-2 spike protein subunits to stimulate macropinocytosis in human alveolar epithelial cells and primary human and murine macrophages. Flow cytometry analysis of fluid-phase marker internalization demonstrated that SARS-CoV-2 spike protein subunits S1, the receptor-binding domain (RBD) of S1, and S2 stimulate macropinocytosis in both human and murine macrophages in an angiotensin-converting enzyme 2 (ACE2)-independent manner. Pharmacological and genetic inhibition of macropinocytosis substantially decreased spike-protein-induced fluid-phase marker internalization in macrophages both in vitro and in vivo. High-resolution scanning electron microscopy (SEM) imaging confirmed that spike protein subunits promote the formation of membrane ruffles on the dorsal surface of macrophages. Mechanistic studies demonstrated that SARS-CoV-2 spike protein stimulated macropinocytosis via NADPH oxidase 2 (Nox2)-derived reactive oxygen species (ROS) generation. In addition, inhibition of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3K) in macrophages blocked SARS-CoV-2 spike-protein-induced macropinocytosis. To our knowledge, these results demonstrate for the first time that SARS-CoV-2 spike protein subunits stimulate macropinocytosis in macrophages. These results may contribute to a better understanding of SARS-CoV-2 infection and COVID-19 pathogenesis.

Identification of a highly conserved neutralizing epitope within the RBD region of diverse SARS-CoV-2 variants

The constant emergence of SARS-CoV-2 variants continues to impair the efficacy of existing neutralizing antibodies, especially XBB.1.5 and EG.5, which showed exceptional immune evasion properties. Here, we identify a highly conserved neutralizing epitope targeted by a broad-spectrum neutralizing antibody BA7535, which demonstrates high neutralization potency against not only previous variants, such as Alpha, Beta, Gamma, Delta and Omicron BA.1-BA.5, but also more recently emerged Omicron subvariants, including BF.7, CH.1.1, XBB.1, XBB.1.5, XBB.1.9.1, EG.5. Structural analysis of the Omicron Spike trimer with BA7535-Fab using cryo-EM indicates that BA7535 recognizes a highly conserved cryptic receptor-binding domain (RBD) epitope, avoiding most of the mutational hot spots in RBD. Furthermore, structural simulation based on the interaction of BA7535-Fab/RBD complexes dissects the broadly neutralizing effect of BA7535 against latest variants. Therapeutic and prophylactic treatment with BA7535 alone or in combination with BA7208 protected female mice from the circulating Omicron BA.5 and XBB.1 variant infection, suggesting the highly conserved neutralizing epitope serves as a potential target for developing highly potent therapeutic antibodies and vaccines.

Interaction analysis of SARS-CoV-2 omicron BA1 and BA2 of RBD with fifty monoclonal antibodies: Molecular dynamics approach

This report provides detailed insights into the interaction of fifty monoclonal antibodies with two recent Omicron variants, BA1 and BA2. It has been observed that numerous mutations in the receptor binding domain (RBD) result in significant structural changes in Omicron, enhancing its ability to mediate viral infections compared to other variants of concern. The following antibodies, namely JX3S304, 7KMG, 7CH4, 7BELCOVOX45, 7CDJ, 7C01, 7JX3S2H14, 6XCA, 7CDI, 7JMO, 7B3O, 6ZER, 6XC7CR3022, JX3S309, 6XC7CC123, 7CM4, 7KMI, 7L7EAZD8895, exhibit a superior binding affinity towards the Spike when compared to the reference CR3022. Four best-docked systems were subjected to further testing through molecular dynamics (MD) simulations. The MM/GBSA free energy for the top-scored complexes of BA1 variant are BA1_JX3S3O4, BA1_7KMI, BA1_7CH4, and BA1_7KMG, with respective values of −56.120 kcal/mol, −41.30 kcal/mol, −17.546 kcal/mol, and −8.527 kcal/mol; and of BA2 variant are BA2_JX3S3O4, BA2_7CM4, BA2_KMG, and BA2_7CH4, with respective values of −40.903 kcal/mol, −23.416 kcal/mol, −17.350 kcal/mol, and −5.460 kcal/mol. Detailed structural/energetic parameters, principal component analysis, and free energy landscape (FEL) studies reveal a significant decrease in antibody resistance due to the disappearance of numerous hydrogen bond interactions and various metastable states. We believe that these crucial mechanistic insights will contribute to breakthroughs in SARS-CoV-2 research.

Analysis of Differences in Lymphocyte Subsets and CD4+ T Cells Immune Activation Between Re-detectable Positive and Non-re-detectable Positive COVID-19 Convalescent Patients

Background: When coronavirus disease 2019 (COVID-19) first broke out, much literature was reported on the re-detectable positive phenomenon of COVID-19 patients during recovery; however, there were few studies on the lymphocyte subsets, T-lymphocyte activation indicators, and levels of specific antibodies between the re-detectable positive (RP) and non-re-detectable positive (NRP) patients. Objectives: This study aimed to analyze the immunological characteristics of RP and NRP patients among convalescent patients from post-discharge COVID-19 patients and to explore immunological factors related to RP. Methods: Anticoagulated whole blood samples were collected from 11 healthy controls (HCs) and 66 COVID-19 convalescent patients. Then, the percentage of lymphocyte subsets and CD4+CD38+/HLA-DR+ T cells were tested with flow cytometry, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) anti-spike protein receptor-binding domain immunoglobulin G (S-RBD-IgG) antibody was detected by chemiluminescence. Results: B cells (%) in the RP group were significantly lower than that in the HC group (P = 0.014), and B cells (%) decreased successively in HC, NRP, and RP groups, with significant differences among the three groups (P = 0.016). CD3+ and CD8+ T cells (%) in the RP group were noticeably higher than that in the NRP group (P = 0.004 and 0.019, respectively); nevertheless, there was no difference in CD4+ T cells (%) and natural killer (NK) cells (%) among the three groups. The CD4+CD38+ and CD4+HLA-DR+ T cells (%) in the RP group were noticeably higher than that in the HC group (P = 0.013 and 0.025). The analysis of differences among the three groups showed that CD4+CD38+ and HLA-DR+ T cells (%) were also significantly different (P = 0.037 and 0.015), and CD4+HLA-DR+ T cells (%) in the three groups increased in turn. Meanwhile, there was a positive correlation between RBD-IgG titer and CD4+HLA-DR+ (%) (P = 0.003, r = 0.517), and the RBD-IgG titer of HLA-DR+ high group was higher than that of HLA-DR+ Low group (P = 0.005) in the COVID-19 convalescent patients. Conclusions: In this study, the immunological characteristics of the RP patients of COVID-19 convalescent patients were analyzed based on the lymphocyte subsets and the relationship between S-RBD-IgG and activated CD4+ T cells. The results showed that the low B cells (%) and the increased CD4+HLA-DR+ T cells (%) in the convalescent patients of COVID-19 might be related to the RP phenomenon, and the activated CD4+ T cells might play a crucial role in specific antibody responses.

Heterologous Sarbecovirus Receptor Binding Domains as Scaffolds for SARS-CoV-2 Receptor Binding Motif Presentation.

Structure-guided rational immunogen design can generate optimized immunogens that elicit a desired humoral response. Design strategies often center on targeting conserved sites on viral glycoproteins that will ultimately confer potent neutralization. For SARS-CoV-2 (SARS-2), the surface-exposed spike glycoprotein includes a broadly conserved portion, the receptor binding motif (RBM), that is required to engage the host cellular receptor, ACE2. Expanding humoral responses to this site may result in a more potent neutralizing antibody response against diverse sarbecoviruses. Here, we used a "resurfacing" approach and iterative design cycles to graft the SARS-2 RBM onto heterologous sarbecovirus scaffolds. The scaffolds were selected to vary the antigenic distance relative to SARS-2 to potentially focus responses to RBM. Multimerized versions of these immunogens elicited broad neutralization against sarbecoviruses in the context of preexisting SARS-2 immunity. These validated engineering approaches can help inform future immunogen design efforts for sarbecoviruses and are generally applicable to other viruses.

Design of human ACE2 mimic miniprotein binders that interact with RBD of SARS-CoV-2 variants of concerns

The world of medicine demands from the research community solutions to the emerging problem of SARS-CoV-2 variants and other such potential global pandemics. With advantages of specificity over small molecule drugs and designability over antibodies, miniprotein therapeutics offers a unique solution to the threats of rapidly emerging SARS-CoV-2 variants. Unfortunately, most of the promising miniprotein binders are de novo designed and it is not viable to generate molecules for each new variant. Therefore in this study, we demonstrate a method for design of miniprotein mimics from the interaction interphase of human angiotensin converting enzyme 2 (ACE2). ACE2 is the natural interacting partner for the SARS-CoV-2 spike receptor binding domain (RBD) and acts as a recognition molecule for viral entry into the host cells. Starting with ACE2 N-terminal triple helix interaction interphase, we generated more than 70 miniprotein sequences. Employing Rosetta folding and docking scores we selected 10 promising miniprotein candidates amongst which 3 were found to be soluble in lab studies. Further, using molecular mechanics (MM) calculations on molecular dynamics (MD) trajectories we test interaction of miniproteins with RBD from various variants of concern (VOC). Presently, we report two key findings; miniproteins in this study are generated using less than 10 lab testing experiments, yet when tested through in-vitro experiments, they show submicro to nanomolar affinities towards SARS-CoV-2 RBD. Also in simulation studies, when compared with previously developed therapeutics, our miniproteins display remarkable ability to mimic ACE2 interphase; making them an ideal solution to the ever evolving problem of VOCs. Communicated by Ramaswamy H. Sarma

Rapid Profiling of the Glycosylation Effects on the Binding of SARS-CoV-2 Spike Protein to Angiotensin-Converting Enzyme 2 Using MALDI-MS with High Mass Detection.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 binds directly to angiotensin-converting enzyme 2 (ACE2), mediating the host cell entry of SARS-CoV-2. Both spike protein and ACE2 are highly glycosylated, which can regulate the binding. Here, we utilized high-mass MALDI-MS with chemical cross-linking for profiling the glycosylation effects on the binding between RBD and ACE2. Overall, it was found that ACE2 glycosylation affects the binding more strongly than does RBD glycosylation. The binding affinity was improved after desialylation or partial deglycosylation (N690) of ACE2, while it decreased after degalactosylation. ACE2 can form dimers in solution, which bind more tightly to the RBD than the ACE2 monomers. The ACE2 dimerization and the binding of RBD to dimeric ACE2 can also be improved by the desialylation or deglycosylation of ACE2. Partial deglycosylation of ACE2 increased the dimerization of ACE2 and the binding affinity of RBD and ACE2 by more than a factor of 2, suggesting its high potential for neutralizing SARS-CoV-2. The method described in the work provided a simple way to analyze the protein-protein interaction without sample purification. It can be widely used for rapid profiling of glycosylation effects on protein-protein interaction for glycosylation-related diseases and the study of multiple interactions between protein and protein aggregates in a single system.

Caveats of chimpanzee ChAdOx1 adenovirus-vectored vaccines to boost anti-SARS-CoV-2 protective immunity in mice

Several COVID-19 vaccines use adenovirus vectors to deliver the SARS-CoV-2 spike (S) protein. Immunization with these vaccines promotes immunity against the S protein, but against also the adenovirus itself. This could interfere with the entry of the vaccine into the cell, reducing its efficacy. Herein, we evaluate the efficiency of an adenovirus-vectored vaccine (chimpanzee ChAdOx1 adenovirus, AZD1222) in boosting the specific immunity compared to that induced by a recombinant receptor-binding domain (RBD)-based vaccine without viral vector. Mice immunized with the AZD1222 human vaccine were given a booster 6 months later, with either the homologous vaccine or a recombinant vaccine based on RBD of the delta variant, which was prevalent at the start of this study. A significant increase in anti-RBD antibody levels was observed in rRBD-boosted mice (31–61%) compared to those receiving two doses of AZD1222 (0%). Significantly higher rates of PepMix™- or RBD-elicited proliferation were also observed in IFNγ-producing CD4 and CD8 cells from mice boosted with one or two doses of RBD, respectively. The lower efficiency of the ChAdOx1-S vaccine in boosting specific immunity could be the result of a pre-existing anti-vector immunity, induced by increased levels of anti-adenovirus antibodies found both in mice and humans. Taken together, these results point to the importance of avoiding the recurrent use of the same adenovirus vector in individuals with immunity and memory against them. It also illustrates the disadvantages of ChAdOx1 adenovirus-vectored vaccine with respect to recombinant protein vaccines, which can be used without restriction in vaccine-booster programs.Key points• ChAdOx1 adenovirus vaccine (AZD1222) may not be effective in boosting anti-SARS-CoV-2 immunity• A recombinant RBD protein vaccine is effective in boosting anti-SARS-CoV-2 immunity in mice• Antibodies elicited by the rRBD-delta vaccine persisted for up to 3 months in mice

SARS-CoV-2 Specific Nanobodies Neutralize Different Variants of Concern and Reduce Virus Load in the Brain of h-ACE2 Transgenic Mice.

Since the beginning of the COVID-19 pandemic, there has been a significant need to develop antivirals and vaccines to combat the disease. In this work, we developed llama-derived nanobodies (Nbs) directed against the receptor binding domain (RBD) and other domains of the Spike (S) protein of SARS-CoV-2. Most of the Nbs with neutralizing properties were directed to RBD and were able to block S-2P/ACE2 interaction. Three neutralizing Nbs recognized the N-terminal domain (NTD) of the S-2P protein. Intranasal administration of Nbs induced protection ranging from 40% to 80% after challenge with the WA1/2020 strain in k18-hACE2 transgenic mice. Interestingly, protection was associated with a significant reduction in virus replication in nasal turbinates and a reduction in virus load in the brain. Employing pseudovirus neutralization assays, we identified Nbs with neutralizing capacity against the Alpha, Beta, Delta, and Omicron variants, including a Nb capable of neutralizing all variants tested. Furthermore, cocktails of different Nbs performed better than individual Nbs at neutralizing two Omicron variants (B.1.529 and BA.2). Altogether, the data suggest the potential of SARS-CoV-2 specific Nbs for intranasal treatment of COVID-19 encephalitis.

Cross-protective HCoV immunity reduces symptom development during SARS-CoV-2 infection.

Numerous clinical parameters link to severe coronavirus disease 2019, but factors that prevent symptomatic disease remain unknown. We investigated the impact of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and endemic human coronavirus (HCoV) antibody responses on symptoms in a longitudinal children cohort (n = 2,917) and a cross-sectional cohort including children and adults (n = 882), all first exposed to SARS-CoV-2 (March 2020 to March 2021) in Switzerland. Saliva (n = 4,993) and plasma (n = 7,486) antibody reactivity to the four HCoVs (subunit S1 [S1]) and SARS-CoV-2 (S1, receptor binding domain, subunit S2 [S2], nucleocapsid protein) was determined along with neutralizing activity against SARS-CoV-2 Wuhan, Alpha, Delta, and Omicron (BA.2) in a subset of individuals. Inferred recent SARS-CoV-2 infection was associated with a strong correlation between mucosal and systemic SARS-CoV-2 anti-spike responses. Individuals with pre-existing HCoV-S1 reactivity exhibited significantly higher antibody responses to SARS-CoV-2 in both plasma (IgG regression coefficients = 0.20, 95% CI = [0.09, 0.32], P < 0.001) and saliva (IgG regression coefficient = 0.60, 95% CI = [0.088, 1.11], P = 0.025). Saliva neutralization activity was modest but surprisingly broad, retaining activity against Wuhan (median NT50 = 32.0, 1Q-3Q = [16.4, 50.2]), Alpha (median NT50 = 34.9, 1Q-3Q = [26.0, 46.6]), and Delta (median NT50 = 28.0, 1Q-3Q = [19.9, 41.7]). In line with a rapid mucosal defense triggered by cross-reactive HCoV immunity, asymptomatic individuals presented with higher pre-existing HCoV-S1 activity in plasma (IgG HKU1, odds ratio [OR] = 0.53, 95% CI = [0.29,0.97], P = 0.038) and saliva (total HCoV, OR = 0.55, 95% CI = [0.33, 0.91], P = 0.019) and higher SARS-CoV-2 reactivity in saliva (IgG S2 fold change = 1.26, 95% CI = [1.03, 1.54], P = 0.030). By investigating the systemic and mucosal immune responses to SARS-CoV-2 and HCoVs in a population without prior exposure to SARS-CoV-2 or vaccination, we identified specific antibody reactivities associated with lack of symptom development.IMPORTANCEKnowledge of the interplay between human coronavirus (HCoV) immunity and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection is critical to understanding the coexistence of current endemic coronaviruses and to building knowledge potential future zoonotic coronavirus transmissions. This study, which retrospectively analyzed a large cohort of individuals first exposed to SARS-CoV-2 in Switzerland in 2020-2021, revealed several key findings. Pre-existing HCoV immunity, particularly mucosal antibody responses, played a significant role in improving SARS-CoV-2 immune response upon infection and reducing symptoms development. Mucosal neutralizing activity against SARS-CoV-2, although low in magnitude, retained activity against SARS-CoV-2 variants underlining the importance of maintaining local mucosal immunity to SARS-CoV-2. While the cross-protective effect of HCoV immunity was not sufficient to block infection by SARS-CoV-2, the present study revealed a remarkable impact on limiting symptomatic disease. These findings support the feasibility of generating pan-protective coronavirus vaccines by inducing potent mucosal immune responses.

Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.

P1056 T cell responses to repeated SARS-CoV-2 vaccination and breakthrough infections in patients with inflammatory bowel disease on TNF inhibitor treatment, a prospective cohort study

Abstract Background Understanding T cell responses to SARS-CoV-2 immunisation in patients on immunosuppressive treatment is important for future vaccine recommendations. Our aim was to investigate the magnitude and quality of T cell responses following repeated SARS-CoV-2 vaccine doses and COVID-19 (breakthrough infections). Methods In this prospective, observational study of responses to SARS-CoV-2 vaccines (Nor-vaC)1, patients with inflammatory bowel disease (IBD) on TNF inhibitor (TNFi) therapy receiving up to four SARS-CoV-2 vaccine doses, and healthy controls receiving three vaccine doses were included. CD4 and CD8 T cell responses to SARS-CoV-2 peptides were measured by flow cytometry before and 2-4 weeks following vaccination and breakthrough infection. Antibodies to the receptor binding domain (RBD) of the Wuhan-Hu-1 and BA.1 (omicron) strains were measured. Results Between March 2, 2021, and December 20, 2022, 118 IBD patients, and 21 healthy controls were included in the study (Table). Following three vaccine doses, the humoral immune response was attenuated in IBD patients compared to healthy controls (median 3954 BAU/ml vs. 6280 BAU/ml, p=0.008). However, both the percentage of spike-specific T cells (CD4 median 0.11% vs. 0.10%, p=0.259, CD8 0.030% vs. 0.046%, p=0,333) and T cell polyfunctionality scores (a measure of quality of vaccine response) (CD4 median 0.403 vs. 0.371, p=0.386, CD8 0.105 vs. 0.101, p=0.871), showed comparable quality in IBD patients and controls following three doses. CD4 T cell responses increased rapidly and plateaued after two vaccine doses whereas CD8 T cell responses continued to improve up to four vaccine doses (Figure). Breakthrough infection in patients following the third or fourth vaccine dose increased CD8 polyfunctionality and generated broad CD4 and CD8 T cell responses against non-spike peptides. There was no difference in T cell responses between patients using TNFi mono- and combo therapy after primary vaccination (three vaccine doses) (p=0.648). Conclusion Despite attenuated antibody responses, IBD patients on TNFi treatment demonstrated T cell responses comparable to healthy controls following three SARS-CoV-2 vaccine doses. Repeated vaccination and breakthrough infection increased the quality and breadth of T cell responses, reaching a plateau. Distinguishing between CD4 and CD8 immune compartments and dynamics of activation after SARS-CoV-2 immunisation may provide nuanced understanding of protective cellular responses The study results indicate that IBD patients on TNFi treatment may in future follow the same recommendations for further SARS-CoV-2 vaccines as the general population. 1Clinialtrials.gov NCT04798625

A RaPID Response to SARS‐CoV‐2

AbstractGenetically encoded peptide libraries are at the forefront of de novo drug discovery. The RaPID (Random Nonstandard Peptides Integrated Discovery) platform stands out due to the unique combination of flexible in vitro translation (FIT) and mRNA display. This enables the incorporation of non‐canonical amino acids, improving chemical diversity and allowing macrocyclisation of the peptide library. The resulting constrained peptides are valued for their strong binding affinity and stability, especially in the context of protein‐protein interactions. In response to SARS‐CoV‐2, the causative agent of the COVID‐19 pandemic, the RaPID system proved valuable in identifying high‐affinity ligands of viral proteins. Among many peptide ligands of SARS‐CoV‐2 spike and main protease (Mpro), several macrocycles stand out for their exceptional binding affinities. Structural data showcases distinct binding modes in complex with the receptor‐binding domain (RBD) of the spike glycoprotein or the catalytic active site of Mpro. However, translating these in vitro findings into clinical applications remains challenging, especially due to insufficient cell permeability.

Standardised quantitative assays for anti-SARS-CoV-2 immune response used in vaccine clinical trials by the CEPI Centralized Laboratory Network: a qualification analysis

Accurate quantitation of immune markers is crucial for ensuring reliable assessment of vaccine efficacy against infectious diseases. This study was designed to confirm standardised performance of SARS-CoV-2 assays used to evaluate COVID-19 vaccine candidates at the initial seven laboratories (in North America, Europe, and Asia) of the Coalition for Epidemic Preparedness Innovations (CEPI) Centralized Laboratory Network (CLN). Three ELISAs (pre-spike protein, receptor binding domain, and nucleocapsid), a microneutralisation assay (MNA), a pseudotyped virus-based neutralisation assay (PNA), and an IFN-γ T-cell ELISpot assay were developed, validated or qualified, and transferred to participating laboratories. Immune responses were measured in ELISA laboratory units (ELU) for ELISA, 50% neuralisation dilution (ND50) for MNA, 50% neutralisation titre (NT50) for PNA, and spot-forming units for the ELISpot assay. Replicate assay results of well characterised panels and controls of blood samples from individuals with or without SARS-CoV-2 infection were evaluated by geometric mean ratios, standard deviation, linear regression, and Spearman correlation analysis for consistency, accuracy, and linearity of quantitative measurements across all laboratories. High reproducibility of results across all laboratories was demonstrated, with interlaboratory precision of 4·1-7·7% coefficient of variation for all three ELISAs, 3·8-19·5% for PNA, and 17·1-24·1% for MNA, over a linear range of 11-30 760 ELU per mL for the three ELISAs, 14-7876 NT50 per mL for PNA, and 21-25 587 ND50 per mL for MNA. The MNA was also adapted for detection of neutralising antibodies against the major SARS-CoV-2 variants of concern. The results of PNA and MNA (r=0·864) and of ELISA and PNA (r=0·928) were highly correlated. The IFN-γ ELISpot interlaboratory variability was 15·9-49·9% coefficient of variation. Sensitivity and specificity were close to 100% for all assays. The CEPI CLN provides accurate quantitation of anti-SARS-CoV-2 immune response across laboratories to allow direct comparisons of different vaccine formulations in different geographical areas. Lessons learned from this programme will serve as a model for faster responses to future pandemic threats and roll-out of effective vaccines. CEPI.

L-Glycosidase-Cleavable Natural Glycans Facilitate the Chemical Synthesis of Correctly Folded Disulfide-Bonded D-Proteins.

D-peptide ligands can be screened for therapeutic potency and enzymatic stability using synthetic mirror-image proteins (D-proteins), but efficient acquisition of these D-proteins can be hampered by the need to accomplish their in vitro folding, which often requires the formation of correctly linked disulfide bonds. Here, we report the finding that temporary installation of natural O-linked-β-N-acetyl-D-glucosamine (O-GlcNAc) groups onto selected D-serine or D-threonine residues of the synthetic disulfide-bonded D-proteins can facilitate their folding in vitro, and that the natural glycosyl groups can be completely removed from the folded D-proteins to afford the desired chirally inverted D-protein targets using naturally occurring O-GlcNAcase. This approach enabled the efficient chemical syntheses of several important but difficult-to-fold D-proteins incorporating disulfide bonds including the mirror-image tumor necrosis factor alpha (D-TNFα) homotrimer and the mirror-image receptor-binding domain of the Omicron spike protein (D-RBD). Our work establishes the use of O-GlcNAc to facilitate D-protein synthesis and folding and proves that D-proteins bearing O-GlcNAc can be good substrates for naturally occurring O-GlcNAcase.