Breakthrough Infections in SARS-CoV-2-Vaccinated Multiple Myeloma Patients Improve Cross-Protection against Omicron Variants.

Patients with multiple myeloma (MM) are a heterogenous, immunocompromised group with increased risk for COVID-19 morbidity and mortality but impaired responses to primary mRNA SARS-CoV-2 vaccination. The effects of booster vaccinations and breakthrough infections (BTIs) on antibody (Ab) levels and cross-protection to variants of concern (VOCs) are, however, not sufficiently evaluated. Therefore, we analysed humoral and cellular vaccine responses in MM patients stratified according to disease stage/treatment into group (1) monoclonal gammopathy of undetermined significance, (2) after stem cell transplant (SCT) without immunotherapy (IT), (3) after SCT with IT, and (4) progressed MM, and in healthy subjects (prospective cohort study). In contrast to SARS-CoV-2 hu-1-specific Ab levels, Omicron-specific Abs and their cross-neutralisation capacity remained low even after three booster doses in a majority of MM patients. In particular, progressed MM patients receiving anti-CD38 mAb and those after SCT with IT were Ab low responders and showed delayed formation of spike-specific B memory cells. However, MM patients with hybrid immunity (i.e., vaccination and breakthrough infection) had improved cross-neutralisation capacity against VOCs, yet in the absence of severe COVID-19 disease. Our results indicate that MM patients require frequent variant-adapted booster vaccinations and/or changes to other vaccine formulations/platforms, which might have similar immunological effects as BTIs.

Identification of a Synthetic Polyhydroxyphenolic Resveratrol Analogue, 3,3',4,4',5,5'-Hexahydroxy-trans-Stilbene with Anti-SARS-CoV-2 Activity.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus has been causing the COVID-19 pandemic since December 2019, with over 600 million infected persons worldwide and over six million deaths. We investigated the anti-viral effects of polyphenolic green tea ingredients and the synthetic resveratrol analogue 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (HHS), a compound with antioxidant, antitumor and anti-HIV properties. In the TCID50 assay, four out of nine green tea constituents showed minor to modest cell protective effects, whereas HHS demonstrated the highest reduction (1103-fold) of the TCID50, indicating pronounced inhibition of virus replication. HHS was also a highly effective inhibitor of SARS-CoV-2 proliferation in VeroE6 cells with an IC50 value of 31.1 µM. HSS also inhibited the binding of the receptor-binding domain (RBD) of the spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor (RBD-ACE2) binding with 29% at 100 µM and with 9.2% at 50 µM indicating that the SARS-CoV-2 inhibitory effect might at least in part be attributed to the inhibition of virus binding to ACE2. Based on the chemical similarity to other polyphenols, the oral bioavailability of HHS is likely also very low, resulting in blood levels far below the inhibitory concentration of EGCG against SARS-CoV-2 observed in vitro. However, administration of HHS topically as a nose or throat spray would increase concentrations several-fold above the minimal inhibitory concentration (MIC) in the mucosa and might reduce virus load when administered soon after infection. Due to these promising tissue culture results, further preclinical and clinical studies are warranted to develop HHS as an additional treatment option for SARS-CoV-2 infection to complement vaccines, which is and will be the main pillar to combat the COVID-19 pandemic.

Inhibition of SARS-CoV-2 Omicron BA.1 and BA.4 Variants After Fourth Vaccination or Tixagevimab and Cilgavimab Administration in Patients With Cancer.

This cohort study assesses the capacity of passive immunization and tixagevimab and cilgavimab to inhibit interaction between receptor-binding domains and angiotensin-converting enzyme 2 in patients with hemato-oncologic diseases.

Art v 1 IgE epitopes of patients and humanized mice are conformational.

BACKGROUND: Worldwide, pollen of the weed mugwort (Artemisiavulgaris) is a major cause of severe respiratory allergy, with its major allergen, Art v 1, being the key pathogenic molecule for millions of patients. Humanized mice transgenic for a human T-cell receptor specific for the major Art v 1 T-cell epitope and the corresponding HLA have been made. OBJECTIVE: We sought to characterize IgE epitopes of Art v 1-sensitized patients and humanized mice for molecular immunotherapy of mugwort allergy. METHODS: Four overlapping peptides incorporating surface-exposed amino acids representing the full-length Art v 1 sequence were synthesized and used to search for IgE reactivity to sequential epitopes. For indirect mapping, peptide-specific rabbit antibodies were raised to block IgE against surface-exposed epitopes on folded Art v 1. IgE reactivity and basophil activation studies were performed in clinically defined mugwort-allergic patients. Secondary structure of recombinant (r) Art v 1 and peptides was determined by circular dichroism spectroscopy. RESULTS: Mugwort-allergic patients and humanized mice sensitized by allergen inhalation showed IgE reactivity and/or basophil activation mainly to folded, complete Art v 1 but not to unfolded, sequential peptide epitopes. Blocking of allergic patients' IgE with peptide-specific rabbit antisera identified a hitherto unknown major conformational IgE binding site in the C-terminal Art v 1 domain. CONCLUSIONS: Identification of the new major conformational IgE binding site on Art v 1, which can be blocked with IgG raised against non-IgE reactive Art v 1 peptides, is an important basis for the development of a hypoallergenic peptide vaccine for mugwort allergy.

Lack of Induction of RBD-Specific Neutralizing Antibodies despite Repeated Heterologous SARS-CoV-2 Vaccination Leading to Seroconversion and Establishment of T Cell-Specific Memory in a Patient in Remission of Multiple Myeloma.

BACKGROUND: Prophylactic vaccination against infectious diseases may induce a state of long-term protection in the otherwise healthy host. However, the situation is less predictable in immunocompromised patients and may require adjustment of vaccination schedules and/or basic therapy. METHODS: A patient in full remission of multiple myeloma since the last three years and on long-term maintenance therapy with pomalidomide, a drug inhibiting angiogenesis and myeloma cell growth, was vaccinated twice with Comirnaty followed by two vaccinations with Vaxzevria. Seroconversion and SARS-CoV-2-specific cellular responses were monitored. RESULTS: No signs of seroconversion or T cellular memory were observed after the first "full immunization" with Comirnaty. Consequently, long-term-maintenance therapy with Pomalidomide was stopped and two additional shots of Vaxzevria were administered after which the patient seroconverted with Spike(S)-protein specific antibody levels reaching 49 BAU/mL, mild S-peptide pool-specific T cell proliferation, effector cytokine production (IL-2, IL-13), and T cellular activation with increased numbers of CD3+CD4+CD25+ T cells as compared to vaccinated and non-vaccinated control subjects. However, despite suspension of immunosuppression and administration of in total four consecutive heterologous SARS-CoV-2 vaccine shots, the patient did not develop neutralizing RBD-specific antibodies. CONCLUSIONS: Despite immunomonitoring-based adjustment of vaccination and/or therapy schedules vaccination success, with clear correlates of protection, the development of RBD-specific antibodies could not be achieved in the immunocompromised patient with current SARS-CoV-2 vaccines. Thus, our report emphasizes the need for improved active and passive immunization strategies for SARS-CoV-2 infections.

The Instability of Dimeric Fc-Fusions Expressed in Plants Can Be Solved by Monomeric Fc Technology.

The potential therapeutic value of many proteins is ultimately limited by their rapid in vivo clearance. One strategy to limit clearance by metabolism and excretion, and improving the stability of therapeutic proteins, is their fusion to the immunoglobulin fragment crystallizable region (Fc). The Fc region plays multiple roles in (i) dimerization for the formation of "Y"-shaped structure of Ig, (ii) Fc-mediated effector functions, (iii) extension of serum half-life, and (iv) a cost-effective purification tag. Plants and in particular Nicotiana benthamiana have proven to be suitable expression platforms for several recombinant therapeutic proteins. Despite the enormous success of their use for the production of full-length monoclonal antibodies, the expression of Fc-fused therapeutic proteins in plants has shown limitations. Many Fc-fusion proteins expressed in plants show different degrees of instability resulting in high amounts of Fc-derived degradation products. To address this issue, we used erythropoietin (EPO) as a reporter protein and evaluated the efforts to enhance the expression of full-length EPO-Fc targeted to the apoplast of N. benthamiana. Our results show that the instability of the fusion protein is independent from the Fc origin or IgG subclass and from the peptide sequence used to link the two domains. We also show that a similar instability occurs upon the expression of individual heavy chains of monoclonal antibodies and ScFv-Fc that mimic the "Y"-shape of antibodies but lack the light chain. We propose that in this configuration, steric hindrance between the protein domains leads to physical instability. Indeed, mutations of critical residues located on the Fc dimerization interface allowed the expression of fully stable EPO monomeric Fc-fusion proteins. We discuss the limitations of Fc-fusion technology in N. benthamiana transient expression systems and suggest strategies to optimize the Fc-based scaffolds on their folding and aggregation resistance in order to improve the stability.

Silencing of SARS-CoV-2 with modified siRNA-peptide dendrimer formulation.

BACKGROUND: First vaccines for prevention of Coronavirus disease 2019 (COVID-19) are becoming available but there is a huge and unmet need for specific forms of treatment. In this study we aimed to evaluate the anti-SARS-CoV-2 effect of siRNA both in vitro and in vivo. METHODS: To identify the most effective molecule out of a panel of 15 in silico designed siRNAs, an in vitro screening system based on vectors expressing SARS-CoV-2 genes fused with the firefly luciferase reporter gene and SARS-CoV-2-infected cells was used. The most potent siRNA, siR-7, was modified by Locked nucleic acids (LNAs) to obtain siR-7-EM with increased stability and was formulated with the peptide dendrimer KK-46 for enhancing cellular uptake to allow topical application by inhalation of the final formulation - siR-7-EM/KK-46. Using the Syrian Hamster model for SARS-CoV-2 infection the antiviral capacity of siR-7-EM/KK-46 complex was evaluated. RESULTS: We identified the siRNA, siR-7, targeting SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) as the most efficient siRNA inhibiting viral replication in vitro. Moreover, we showed that LNA-modification and complexation with the designed peptide dendrimer enhanced the antiviral capacity of siR-7 in vitro. We demonstrated significant reduction of virus titer and lung inflammation in animals exposed to inhalation of siR-7-EM/KK-46 in vivo. CONCLUSIONS: Thus, we developed a therapeutic strategy for COVID-19 based on inhalation of a modified siRNA-peptide dendrimer formulation. The developed medication is intended for inhalation treatment of COVID-19 patients.

SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8+ T cell responses.

CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

Prevention of allergy by virus-like nanoparticles (VNP) delivering shielded versions of major allergens in a humanized murine allergy model.

BACKGROUND: In high-risk populations, allergen-specific prophylaxis could protect from sensitization and subsequent development of allergic disease. However, such treatment might itself induce sensitization and allergies, thus requiring hypoallergenic vaccine formulations. We here characterized the preventive potential of virus-like nanoparticles (VNP) expressing surface-exposed or shielded allergens. METHODS: Full-length major mugwort pollen allergen Art v 1 was selectively targeted either to the surface or to the inner side of the lipid bilayer envelope of VNP. Upon biochemical and immunological analysis, their preventive potential was determined in a humanized mouse model of mugwort pollen allergy. RESULTS: Virus-like nanoparticles expressing shielded version of Art v 1, in contrast to those expressing surface-exposed Art v 1, were hypoallergenic as they hardly induced degranulation of rat basophil leukemia cells sensitized with Art v 1-specific mouse or human IgE. Both VNP versions induced proliferation and cytokine production of allergen-specific T cells in vitro. Upon intranasal application in mice, VNP expressing surface-exposed but not shielded allergen induced allergen-specific antibodies, including IgE. Notably, preventive treatment with VNP expressing shielded allergen-protected mice from subsequent sensitization with mugwort pollen extract. Protection was associated with a Th1/Treg-dominated cytokine response, increased Foxp3+ Treg numbers in lungs, and reduced lung resistance when compared to mice treated with empty particles. CONCLUSION: Virus-like nanoparticles represent a novel and versatile platform for the in vivo delivery of allergens to selectively target T cells and prevent allergies without inducing allergic reactions or allergic sensitization.

Characterization of plants expressing the human ß1,4-galactosyltrasferase gene.

Modification of the plant N-glycosylation pathway towards human type structures is an important strategy to implement plants as expression systems for therapeutic proteins. Nevertheless, relatively little is known about the overall impact of non-plant glycosylation enzymes in stable transformed plants. Here, we analyzed transgenic lines (Nicotiana benthamiana and Arabidopsis thaliana) that stably express a modified version of human ß1,4-galactosyltransferase ((ST)GalT). While some transgenic plants grew normally, other lines exhibited a severe phenotype associated with stunted growth and developmental retardation. The severity of the phenotype correlated with both increased (ST)GalT mRNA and protein levels but no differences were observed between N-glycosylation profiles of plants with and without the phenotype. In contrast to non-transgenic plants, all (ST)GalT expressing plants synthesized significant amounts of incompletely processed (largely depleted of core fucose) N-glycans with up to 40% terminally galactosylated structures. While transgenic plants showed no differences in nucleotide sugar composition and cell wall monosaccharide content, alterations in the reactivity of cell wall carbohydrate epitopes associated with arabinogalactan-proteins and pectic homogalacturonan were detected in (ST)GalT expressing plants. Notably, plants with phenotypic alterations showed increased levels of hydrogen peroxide, most probably a consequence of hypersensitive reactions. Our data demonstrate that unfavorable phenotypical modifications may occur upon stable in planta expression of non-native glycosyltransferases. Such important issues need to be taken into consideration in respect to stable glycan engineering in plants.

Proteolytic and N-glycan processing of human α1-antitrypsin expressed in Nicotiana benthamiana.

Plants are increasingly being used as an expression system for complex recombinant proteins. However, our limited knowledge of the intrinsic factors that act along the secretory pathway, which may compromise product integrity, renders process design difficult in some cases. Here, we pursued the recombinant expression of the human protease inhibitor α1-antitrypsin (A1AT) in Nicotiana benthamiana. This serum protein undergoes intensive posttranslational modifications. Unusually high levels of recombinant A1AT were expressed in leaves (up to 6 mg g(-1) of leaf material) in two forms: full-length A1AT located in the endoplasmic reticulum displaying inhibitory activity, and secreted A1AT processed in the reactive center loop, thus rendering it unable to interact with target proteinases. We found that the terminal protein processing is most likely a consequence of the intrinsic function of A1AT (i.e. its interaction with proteases [most likely serine proteases] along the secretory pathway). Secreted A1AT carried vacuolar-type paucimannosidic N-glycans generated by the activity of hexosaminidases located in the apoplast/plasma membrane. Notwithstanding, an intensive glycoengineering approach led to secreted A1AT carrying sialylated N-glycan structures largely resembling its serum-derived counterpart. In summary, we elucidate unique insights in plant glycosylation processes and show important aspects of postendoplasmic reticulum protein processing in plants.

Generation of biologically active multi-sialylated recombinant human EPOFc in plants.

Hyperglycosylated proteins are more stable, show increased serum half-life and less sensitivity to proteolysis compared to non-sialylated forms. This applies particularly to recombinant human erythropoietin (rhEPO). Recent progress in N-glycoengineering of non-mammalian expression hosts resulted in in vivo protein sialylation at great homogeneity. However the synthesis of multi-sialylated N-glycans is so far restricted to mammalian cells. Here we used a plant based expression system to accomplish multi-antennary protein sialylation. A human erythropoietin fusion protein (EPOFc) was transiently expressed in Nicotiana benthamiana ΔXTFT, a glycosylation mutant that lacks plant specific N-glycan residues. cDNA of the hormone was co-delivered into plants with the necessary genes for (i) branching (ii) ß1,4-galactosylation as well as for the (iii) synthesis, transport and transfer of sialic acid. This resulted in the production of recombinant EPOFc carrying bi- tri- and tetra-sialylated complex N-glycans. The formation of this highly complex oligosaccharide structure required the coordinated expression of 11 human proteins acting in different subcellular compartments at different stages of the glycosylation pathway. In vitro receptor binding assays demonstrate the generation of biologically active molecules. We demonstrate the in planta synthesis of one of the most complex mammalian glycoforms pointing to an outstanding high degree of tolerance to changes in the glycosylation pathway in plants.

N-glycosylation engineering of plants for the biosynthesis of glycoproteins with bisected and branched complex N-glycans.

Glycoengineering is increasingly being recognized as a powerful tool to generate recombinant glycoproteins with a customized N-glycosylation pattern. Here, we demonstrate the modulation of the plant glycosylation pathway toward the formation of human-type bisected and branched complex N-glycans. Glycoengineered Nicotiana benthamiana lacking plant-specific N-glycosylation (i.e. ß1,2-xylose and core α1,3-fucose) was used to transiently express human erythropoietin (hEPO) and human transferrin (hTF) together with modified versions of human ß1,4-mannosyl-ß1,4-N-acetylglucosaminyltransferase (GnTIII), α1,3-mannosyl-ß1,4-N-acetylglucosaminyltransferase (GnTIV) and α1,6-mannosyl-ß1,6-N-acetylglucosaminyltransferase (GnTV). hEPO was expressed as a fusion to the IgG-Fc domain (EPO-Fc) and purified via protein A affinity chromatography. Recombinant hTF was isolated from the intracellular fluid of infiltrated plant leaves. Mass spectrometry-based N-glycan analysis of hEPO and hTF revealed the quantitative formation of bisected (GnGnbi) and tri- as well as tetraantennary complex N-glycans (Gn[GnGn], [GnGn]Gn and [GnGn][GnGn]). Co-expression of GnTIII together with GnTIV and GnTV resulted in the efficient generation of bisected tetraantennary complex N-glycans. Our results show the generation of recombinant proteins with human-type N-glycosylation at great uniformity. The strategy described here provides a robust and straightforward method for producing mammalian-type N-linked glycans of defined structures on recombinant glycoproteins, which can advance glycoprotein research and accelerate the development of protein-based therapeutics.

In planta protein sialylation through overexpression of the respective mammalian pathway.

Many therapeutic proteins are glycosylated and require terminal sialylation to attain full biological activity. Current manufacturing methods based on mammalian cell culture allow only limited control of this important posttranslational modification, which may lead to the generation of products with low efficacy. Here we report in vivo protein sialylation in plants, which have been shown to be well suited for the efficient generation of complex mammalian glycoproteins. This was achieved by the introduction of an entire mammalian biosynthetic pathway in Nicotiana benthamiana, comprising the coordinated expression of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to terminal galactose. We show the transient overexpression and functional integrity of six mammalian proteins that act at various stages of the biosynthetic pathway and demonstrate their correct subcellular localization. Co-expression of these genes with a therapeutic glycoprotein, a human monoclonal antibody, resulted in quantitative sialylation of the Fc domain. Sialylation was at great uniformity when glycosylation mutants that lack plant-specific N-glycan residues were used as expression hosts. Finally, we demonstrate efficient neutralization activity of the sialylated monoclonal antibody, indicating full functional integrity of the reporter protein. We report for the first time the incorporation of the entire biosynthetic pathway for protein sialylation in a multicellular organism naturally lacking sialylated glycoconjugates. Besides the biotechnological impact of the achievement, this work may serve as a general model for the manipulation of complex traits into plants.

Improved virus neutralization by plant-produced anti-HIV antibodies with a homogeneous beta1,4-galactosylated N-glycan profile.

It is well established that proper N-glycosylation significantly influences the efficacy of monoclonal antibodies (mAbs). However, the specific immunological relevance of individual mAb-associated N-glycan structures is currently largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. Here we report on the generation of a plant-based expression platform allowing the efficient production of mAbs with a homogeneous beta1,4-galactosylated N-glycosylation structure, the major N-glycan species present on serum IgG. This was achieved by the expression of a highly active modified version of the human beta1,4-galactosyltransferase in glycoengineered plants lacking plant-specific glycosylation. Moreover, we demonstrate that two anti-human immunodeficiency virus mAbs with fully beta1,4-galactosylated N-glycans display improved virus neutralization potency when compared with other glycoforms produced in plants and Chinese hamster ovary cells. These findings indicate that mAbs containing such homogeneous N-glycan structures should display improved in vivo activities. Our system, using expression of mAbs in tobacco plants engineered for post-translational protein processing, provides a new means of overcoming the two hurdles that limit the therapeutic use of anti-human immunodeficiency virus mAbs in global health initiatives, low biological potency and high production costs.