Profiling antibody epitopes induced by mRNA-1273 vaccination and boosters.

Background: Characterizing the antibody epitope profiles of messenger RNA (mRNA)-based vaccines against SARS-CoV-2 can aid in elucidating the mechanisms underlying the antibody-mediated immune responses elicited by these vaccines. Methods: This study investigated the distinct antibody epitopes toward the SARS-CoV-2 spike (S) protein targeted after a two-dose primary series of mRNA-1273 followed by a booster dose of mRNA-1273 or a variant-updated vaccine among serum samples from clinical trial adult participants. Results: Multiple S-specific epitopes were targeted after primary vaccination; while signal decreased over time, a booster dose after >6 months largely revived waning antibody signals. Epitope identity also changed after booster vaccination in some subjects, with four new S-specific epitopes detected with stronger signals after boosting than with primary vaccination. Notably, the strength of antibody responses after booster vaccination differed by the exact vaccine formulation, with variant-updated mRNA-1273.211 and mRNA-1273.617.2 booster formulations inducing significantly stronger S-specific signals than a mRNA-1273 booster. Conclusion: Overall, these results identify key S-specific epitopes targeted by antibodies induced by mRNA-1273 primary and variant-updated booster vaccination.

Quantifying the Vaccine-Induced Humoral Immune Response to Spike-Receptor Binding Domain as a Surrogate for Neutralization Testing Following mRNA-1273 (Spikevax) Vaccination Against COVID-19.

INTRODUCTION: There is a need for automated, high-throughput assays to quantify immune response after SARS-CoV-2 vaccination. This study assessed the combined utility of the Elecsys® Anti-SARS-CoV-2 S (ACOV2S) and the Elecsys Anti-SARS-CoV-2 (ACOV2N) assays using samples from the mRNA-1273 (Spikevax™) phase 2 trial (NCT04405076). METHODS: Samples from 593 healthy participants in two age cohorts (18-54 and ≥ 55 years), who received two injections with placebo (n = 198) or mRNA-1273 (50 µg [n = 197] or 100 µg [n = 198]), were collected at days 1 (first vaccination), 15, 29 (second vaccination), 43, and 57. ACOV2S results were used to assess humoral response to vaccination in different subgroups and were compared to live virus microneutralization assay. Samples from patients with either previous or concomitant infection (identified per ACOV2N) were analyzed separately. RESULTS: Receptor-binding domain-specific antibodies were readily detectable by ACOV2S for the vast majority of participants (174/189, 92.1% [50 µg dose] and 178/192, 92.7% [100 µg dose]) at the first post-vaccination assessment, with non-converters predominantly older in age. Seroconversion for all participants was observed at day 29 (before the second vaccine dose). Two weeks after the first dose, geometric mean concentration (GMC) of antibody levels was 1.37-fold higher in the 100 versus 50 µg group (p = 0.0098), reducing to 1.09-fold 2 weeks after the second dose (p = 0.0539, n.s.). In both dose groups, a more pronounced response was observed in the younger versus older age group on day 15 (50 µg, 2.49-fold [p < 0.0001]; 100 µg, 3.94-fold [p < 0.0001] higher GMC, respectively), and day 29 (1.93-fold, p = 0.0002, and 2.44-fold, p < 0.0001). Eight subjects had previous or concomitant SARS-CoV-2 infection; vaccination boosted their humoral response to very high ACOV2S results compared to infection-naïve recipients. ACOV2S strongly correlated with microneutralization (Pearson's r = 0.779; p < 0.0001), including good qualitative agreement. CONCLUSION: These results confirmed that ACOV2S is a highly valuable assay for tracking vaccine-related immune responses. Combined application with ACOV2N enables monitoring for breakthrough infection or stratification of previous natively infected individuals. The adaptive measuring range and high resolution of ACOV2S allow for early identification of seroconversion and resolution of very high titers and longitudinal differences between subgroups. Additionally, good correlation with live virus microneutralization suggests that ACOV2S is a reliable estimate of neutralization capacity in routine diagnostic settings.

mRNA-1273 Vaccine-elicited Neutralization of SARS-CoV-2 Omicron in Adolescents and Children.

Background: The highly transmissible severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Omicron variant is a global concern. This study assessed the neutralization activity of two-dose regimens of mRNA-1273 vaccination against Omicron in adults, adolescents and children. Methods: Neutralizing activity against the Omicron variant was evaluated in serum samples from adults (≥18 years) in the phase 3, Coronavirus Efficacy (COVE) and from adolescents (12-17 years) in the TeenCOVE trials following a two-dose regimen of 100 µg mRNA-1273 and from children (6-<12 years) in the KidCOVE trial administered two doses of 50 µg mRNA-1273. Neutralizing antibody geometric mean ID50 titers (GMT) were measured using a lentivirus-based pseudovirus neutralizing assay at day 1 and 4 weeks (day 57) following the second mRNA-1273 dose, compared with wild-type (D614G). Results: At 4 weeks following a second dose of mRNA-1273 (100 µg), the GMT was reduced 28.8-fold compared with D614G in adults (≥18 years). In adolescents (12-17 years), the GMT was 11.8-fold lower than D614G, 4 weeks after a second dose of mRNA-1273 (100 µg), and compared with adults, were 1.5- and 3.8-fold higher for D614G and the Omicron variant, respectively. In children (6-<12 years), 4 weeks post-second dose of 50 µg mRNA-1273, Omicron GMTs were reduced 22.1-fold versus D614G and were 2.0-fold higher for D614G and 2.5-fold higher for Omicron compared with adults. Conclusions: A two-dose regimen of 100 µg mRNA-1273 in adolescents and of 50 µg in children elicited neutralization responses against the Omicron variant that were reduced compared with the wild-type D614G, and numerically higher than those in adults.

Booster of mRNA-1273 Strengthens SARS-CoV-2 Omicron Neutralization.

The Omicron variant of SARS-CoV-2 is raising concerns because of its increased transmissibility and potential for reduced susceptibility to antibody neutralization. To assess the potential risk of this variant to existing vaccines, serum samples from mRNA-1273 vaccine recipients were tested for neutralizing activity against Omicron and compared to neutralization titers against D614G and Beta in live virus and pseudovirus assays. Omicron was 41-84-fold less sensitive to neutralization than D614G and 5.3-7.4-fold less sensitive than Beta when assayed with serum samples obtained 4 weeks after 2 standard inoculations with 100 µg mRNA-1273. A 50 µg boost increased Omicron neutralization titers and may substantially reduce the risk of symptomatic vaccine breakthrough infections.

Biological evaluation of multivalent lewis X-MGL-1 interactions.

Myeloid C-type lectin receptors (CLRs) expressed by antigen-presenting cells are pattern-recognition receptors involved in the recognition of pathogens as well as of self-antigens. The interaction of carbohydrate ligands with a CLR can trigger immune responses. Although several CLR ligands are known, there is limited insight into CLR targeting by carbohydrate ligands. The weak affinity of lectin-carbohydrate interactions often renders multivalent carbohydrate presentation necessary. Here, we have analyzed the impact of multivalent presentation of the trisaccharide Lewis X (Le(X) ) epitope on its interaction with the CLR macrophage galactose-type lectin-1 (MGL-1). Glycan arrays, including N-glycan structures with terminal Le(X) , were prepared by enzymatic extension of immobilized synthetic core structures with two recombinant glycosyltransferases. Incubation of arrays with an MGL-1-hFc fusion protein showed up to tenfold increased binding to multiantennary N-glycans displaying Le(X) structures, compared to monovalent Le(X) trisaccharide. Multivalent presentation of Le(X) on the model antigen ovalbumin (OVA) led to increased cytokine production in a dendritic cell /T cell coculture system. Furthermore, immunization of mice with Le(X) -OVA conjugates modulated cytokine production and the humoral response, compared to OVA alone. This study provides insights into how multivalent carbohydrate-lectin interactions can be exploited to modulate immune responses.

A platform to screen for C-type lectin receptor-binding carbohydrates and their potential for cell-specific targeting and immune modulation.

Myeloid C-type lectin receptors (CLRs) in innate immunity represent a superfamily of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-antigens. The primary interaction of an antigen-presenting cell and a pathogen shapes the following immune response. Therefore, the identification of CLR ligands that can either enhance or modulate the immune response is of interest. We have developed a screening platform based on glycan arrays to identify immune modulatory carbohydrate ligands of CLRs. A comprehensive library of CLRs was expressed by fusing the extracellular part of each respective CLR, the part containing the carbohydrate-recognition domain (CRD), to the Fc fragment of human IgG1 molecules. CLR-Fc fusion proteins display the CRD in a dimeric form, are properly glycosylated, and can be detected by a secondary antibody with a conjugated fluorophore. Thus, they are valuable tools for high-throughput screening. We were able to identify novel carbohydrate binders of CLRs using the glycan array technology. These CLR-binding carbohydrates were then covalently attached to the model antigen ovalbumin. The ovalbumin neoglycoconjugates were used in a dendritic cell/T cell co-culture assay to stimulate transgenic T cells in vitro. In addition, mice were immunized with these conjugates to analyze the immune modulatory properties of the CLR ligands in vivo. The CLR ligands induced an increased Th1 cytokine production in vitro and modulated the humoral response in vivo. The platform described here allows for the identification of CLR ligands, as well as the evaluation of each ligand's cell-specific targeting and immune modulatory properties.

The C-type lectin receptor DCIR is crucial for the development of experimental cerebral malaria.

Cerebral malaria (CM) is the most severe complication of malaria. The murine Plasmodium berghei ANKA (PbA) infection model has helped to identify crucial players in the pathogenesis of CM. However, the role of pattern recognition receptors in innate immunity to CM induction is still poorly understood. C-type lectin receptors (CLRs) represent a family of pattern recognition receptors that recognize carbohydrate structures on pathogens and self-Ags often in a Ca(2+)-dependent manner. In this study, we investigated the role of the CLR dendritic cell immunoreceptor (DCIR) in the genesis of CM. Using the murine PbA infection, we show in this article that DCIR is essential for the development of CM. Although PbA infection led to 80% CM in wild-type C57BL/6 mice, DCIR-deficient mice were highly protected with only 15% CM development. In accordance with the reduced CM incidence in DCIR(-/-) mice, CD8(+) T cell sequestration was markedly reduced in brains of PbA-infected DCIR(-/-) mice, which was accompanied by reduced brain inflammation. Reduced T cell sequestration in the brain was caused by decreased TNF-α levels in sera, as well as a modulated activation of CD4(+) and CD8(+) T cells in spleen of PbA-infected DCIR(-/-) mice. This study indicates that DCIR is critically involved in CM induction, thus highlighting the importance of this CLR in innate immunity during malaria infection.

Hexameric supramolecular scaffold orients carbohydrates to sense bacteria.

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated ß-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus ß-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.

Design, synthesis and biological evaluation of carbohydrate-functionalized cyclodextrins and liposomes for hepatocyte-specific targeting.

Targeting glycan-binding receptors is an attractive strategy for cell-specific drug and gene delivery. The C-type lectin asialoglycoprotein receptor (ASGPR) is particularly suitable for liver-specific delivery due to its exclusive expression by parenchymal hepatocytes. In this study, we designed and developed an efficient synthesis of carbohydrate-functionalized ß-cyclodextrins (ßCDs) and liposomes for hepatocyte-specific delivery. For targeting of ASGPR, rhodamine B-loaded ßCDs were functionalized with glycodendrimers. Liposomes were equipped with synthetic glycolipids containing a terminal D-GalNAc residue to mediate binding to ASGPR. Uptake studies in the human hepatocellular carcinoma cell line HepG2 demonstrated that ßCDs and liposomes displaying terminal D-Gal/D-GalNAc residues were preferentially endocytosed. In contrast, uptake of ßCDs and liposomes with terminal d-Man or D-GlcNAc residues was markedly reduced. The d-Gal/d-GalNAc-functionalized ßCDs and liposomes presented here enable hepatocyte-specific targeting. Gal-functionalized ßCDs are efficient molecular carriers to deliver doxorubicin in vitro into hepatocytes and induce apoptosis.