Discovery of nanobodies against SARS-CoV-2 and an uncommon neutralizing mechanism

SARS-CoV-2 and its variants continue to threaten public health. The virus recognizes the host cell by attaching its Spike receptor-binding domain (RBD) to the host receptor ACE2. Therefore, RBD is a primary target for neutralizing antibodies and vaccines. Here we report the isolation, and biological and structural characterization of two single-chain antibodies (nanobodies, DL4 and DL28) from RBD-immunized alpaca. Both nanobodies bind Spike with affinities that exceeded the detection limit (picomolar) of the biolayer interferometry assay and neutralize the original SARS-CoV- 2 strain with IC50 of 0.086 g mL-1 (DL4) and 0.385 g mL-1 (DL28). DL4 and a more potent, rationally designed mutant, neutralizes the Alpha variant as potently as the original strain but only displays marginal activity against the Beta variant. By contrast, the neutralizing activity of DL28, when in the Fc-fused divalent form, was less affected by the mutations in the Beta variant (IC50 of 0.414 g mL-1 for Alpha, 1.060 g mL-1 for Beta). Crystal structure studies reveal that DL4 blocks ACE2-binding by direct competition, while DL28 neutralizes SARS-CoV-2 by an uncommon mechanism through which DL28 distorts the receptor-binding motif in RBD and hence prevents ACE2-binding. Our work provides two neutralizing nanobodies for potential therapeutic development and reveals an uncommon mechanism to design and screen novel neutralizing antibodies against SARS-CoV-2.

A high-affinity RBD-targeting nanobody improves fusion partner's potency against SARS-CoV-2

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research have been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and greasy site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.

The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a {beta}-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins. The involvement of each of these proteins and their interplays during the assembly process of this new virus are poorly-defined and are likely {beta}-coronavirus-type different. Therefore, we sought to investigate how SARS-CoV-2 behaves for its assembly by expression assays of S, in combination with E, M and/or N. By combining biochemical and imaging assays, we showed that E and M regulate intracellular trafficking of S and hence its furin-mediated processing. Indeed, our imaging data revealed that S remains at ERGIC or Golgi compartments upon expression of E or M, like for SARS-CoV-2 infected cells. By studying a mutant of S, we showed that its cytoplasmic tail, and more specifically, its C-terminal retrieval motif, is required for the M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlighted that E and M induce a specific maturation of S N-glycosylation, which is observed on particles and lysates from infected cells independently of its mechanisms of intracellular retention. Finally, we showed that both M, E and N are required for optimal production of virus-like-proteins. Altogether, our results indicated that E and M proteins influence the properties of S proteins to promote assembly of viral particles. Our results therefore highlight both similarities and dissimilarities in these events, as compared to other {beta}-coronaviruses. Author SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Its viral particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins, though their involvement in the virion assembly remain unknown for this particular coronavirus. Here we showed that presence of E and M influence the localization and maturation of S protein, in term of cleavage and N-glycosylation maturation. Indeed, E protein is able to slow down the cell secretory pathway whereas M-induced retention of S requires the retrieval motif in S C-terminus. We also highlighted that E and M might regulate the N glycosylation maturation of S independently of its intracellular retention mechanism. Finally, we showed that the four structural proteins are required for optimal formation of virus-like particles, highlighting the involvement of N, E and M in assembly of infectious particles. Altogether, our results highlight both similarities and dissimilarities in these events, as compared to other {beta}-coronaviruses.

Potent synthetic nanobodies against SARS-CoV-2 and molecular basis for neutralization

SARS-CoV-2, the causative agent of COVID-191, recognizes host cells by attaching its receptor-binding domain (RBD) to the host receptor ACE22-7. Neutralizing antibodies that block RBD-ACE2 interaction have been a major focus for therapeutic development8-18. Llama-derived single-domain antibodies (nanobodies, [~]15 kDa) offer advantages including ease of production and possibility for direct delivery to the lungs by nebulization19, which are attractive features for bio-drugs against the global respiratory disease. Here, we generated 99 synthetic nanobodies (sybodies) by in vitro selection using three libraries. The best sybody, MR3 bound to RBD with high affinity (KD = 1.0 nM) and showed high neutralization activity against SARS-CoV-2 pseudoviruses (IC50 = 0.40 g mL-1). Structural, biochemical, and biological characterization of sybodies suggest a common neutralizing mechanism, in which the RBD-ACE2 interaction is competitively inhibited by sybodies. Various forms of sybodies with improved potency were generated by structure-based design, biparatopic construction, and divalent engineering. Among these, a divalent MR3 conjugated with the albumin-binding domain for prolonged half-life displayed highest potency (IC50 = 12 ng mL-1) and protected mice from live SARS-CoV-2 challenge. Our results pave the way to the development of therapeutic nanobodies against COVID-19 and present a strategy for rapid responses for future outbreaks.

Immunization with the receptor-binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement

Recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic. Currently, there is no vaccine available for preventing SARS-CoV-2 infection. Like closely related severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 also uses its receptor-binding domain (RBD) on the spike (S) protein to engage the host receptor, human angiotensin-converting enzyme 2 (ACE2), facilitating subsequent viral entry. Here we report the immunogenicity and vaccine potential of SARS-CoV-2 RBD (SARS2-RBD)-based recombinant proteins. Immunization with SARS2-RBD recombinant proteins potently induced a multi-functional antibody response in mice. The resulting antisera could efficiently block the interaction between SARS2-RBD and ACE2, inhibit S-mediated cell-cell fusion, and neutralize both SARS-CoV-2 pseudovirus entry and authentic SARS-CoV-2 infection. In addition, the anti-RBD sera also exhibited cross binding, ACE2-blockade, and neutralization effects towards SARS-CoV. More importantly, we found that the anti-RBD sera did not promote antibody-dependent enhancement of either SARS-CoV-2 pseudovirus entry or authentic virus infection of Fc receptor-bearing cells. These findings provide a solid foundation for developing RBD-based subunit vaccines for SARS-CoV2.

Progress of immunotherapy in neuromyelitis optica spectrum disorders / 中华全科医师杂志

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune demyelinating disease in central nervous system,mainly involving the optic nerves and spinal cord.NMOSD is a relapsing disease with severe residual disability,therefore,prevention of attacks is critical for the preservation of visual and neurologic function.The conventional immunosuppressants,including prednisone,azathioprine,mycophenolate mofetil and methotrexate,can be used to prevent relapse of NMOSD.And immunotherapeutics such as rituximab,tocilizumab and eculizumab are increasingly used in recent years.This review summarizes the current status and the future strategies in treatment of NMOSD based on the progress of NMOSD pathogenesis.

Effect of vitamin D level on vascular endothelial function and plasma renin activity in patients with hypertension / 中国医师杂志

Objective To analyze the correlation between vitamin D levels and vascular endothelial function and plasma renin activity (PRA) in hypertensive patients.Methods 415 patients with essential hypertension admitted to our hospital from January 1 to December 2017 were selected as the experimental group.142 healthy subjects who received physical examination and met the included indicators were selected as the control group.The blood biochemical indexes and the 25 (OH) D contents in the blood of two groups were detected and analyzed.The experimental group was divided into vitamin D mild deficiency group (15-30 mmol/L),severe deficiency group [25 (OH) D ＜ 15 mmol/L] and normal group [25 (OH)D ＞ 30 mmol/L] according to vitamin D content.The endothelial function (RHI value) and renin were compared in the three groups,and multiple linear regression analysis was performed on endothelial function and orthotopic renin activity and related factors.Results The diastolic blood pressure,vitamin D content and blood lipid biochemical indexes of the experimental group and the control group were significantly different (P ＞ 0.05).The higher the vitamin D content,the lower the plasma renin activity PRA;the RHI value of the vitamin D severe deficiency group was lower than the vitamin D normal group [(1.4 ±0.15)mmol/L vs (1.6 ± 0.20) mmol/L] (P ＜ 0.05).Vitamin D was negatively correlated with 24 hours systolic blood pressure (24 h SSD),the standard deviation of 24 hours diastolic blood pressure (24 h DSD),the Standard deviation of daytime systolic blood pressure (dSSD),the Standard deviation of daytime diastolic blood pressure (dDSD),the Standard deviation of nighttime systolic blood pressure (nSSD),the Standard deviation of nighttime diastolic blood pressure (nDSD) (P ＜ 0.05),and positively correlated with the decrease of night systolic blood pressure and night diastolic blood pressure.Vitamin D and total cholesterol were independent influencing factors of endothelial function (P ＜ 0.01).Vitamin D and 24 h mean heart rate were independent influencing factors of orthostatic renin activity (P ＜ 0.01).Conclusions The low vitamin D content in patients with essential hypertension affects endothelial function and plasma renin activity,which should be paid more attention in clinical practice.

The study on the association of serum interleukin-27 levels with disease severity in neuromyelitis optica / 中国神经精神疾病杂志

Objective To examine the interleukin-27 (IL-27) levels in the serum of the patients with neuromyelitis optica (NMO) and healthy controls (HCs) and explore the correlation of the serum IL-27 level with disease severity.Methods Serum concentrations of IL-27 from 39 patients with NMO and 39 HCs were measured by using enzyme-linked immunosorbent assay (ELISA).Mann-Whitney U test was performed to analyze the difference in the IL-27 levels between the NMO group and the HCs.Spearman's rank correlation analysis was used to analyze the correlations of serum IL-27 levels with clinical parameters (EDSS,spinal cord lesion length,annual relapse rate and antibody titers of AQP-4) of NMO.Results The serum IL-27 levels were significantly lower in NMO group than in HCs (P＜0.001).Serum IL-27 levels were negatively correlated with EDSS,total length of spinal cord lesion identified by MRI at the sampling,and the average relapse rate during two-year follow-up (r=-0.439,P=0.010;r=-0.434,P=0.006;r=-0.451,P=0.031).There was no significant correlation between IL-27 levels and antibody titers of AQP-4 (r=-0.027,P=0.871).Multivariate regression analysis showed that serum IL-27 levels were negatively correlated with EDSS (B=-0.025,P=0.023).Conclusion IL-27 may participate in the pathogenic process of NMO and might be a prognosis marker of the disease.

The study on the association of serum interleukin-27 levels with disease severity in neuromyelitis optica / 中国神经精神疾病杂志

Objective To examine the interleukin-27 (IL-27) levels in the serum of the patients with neuromyelitis optica (NMO) and healthy controls (HCs) and explore the correlation of the serum IL-27 level with disease severity.Methods Serum concentrations of IL-27 from 39 patients with NMO and 39 HCs were measured by using enzyme-linked immunosorbent assay (ELISA).Mann-Whitney U test was performed to analyze the difference in the IL-27 levels between the NMO group and the HCs.Spearman's rank correlation analysis was used to analyze the correlations of serum IL-27 levels with clinical parameters (EDSS,spinal cord lesion length,annual relapse rate and antibody titers of AQP-4) of NMO.Results The serum IL-27 levels were significantly lower in NMO group than in HCs (P＜0.001).Serum IL-27 levels were negatively correlated with EDSS,total length of spinal cord lesion identified by MRI at the sampling,and the average relapse rate during two-year follow-up (r=-0.439,P=0.010;r=-0.434,P=0.006;r=-0.451,P=0.031).There was no significant correlation between IL-27 levels and antibody titers of AQP-4 (r=-0.027,P=0.871).Multivariate regression analysis showed that serum IL-27 levels were negatively correlated with EDSS (B=-0.025,P=0.023).Conclusion IL-27 may participate in the pathogenic process of NMO and might be a prognosis marker of the disease.

miR-15a and miR-16 modulate drug resistance by targeting bcl-2 in human colon cancer cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the reversal effect of targeted modulation of bcl-2 expression by miR-15a and miR-16 on drug resistance of human colon cancer cells.</p><p><b>METHODS</b>Mimics or inhibitors of miR-15a and miR-16 were transfected into HCT8 or HCT8/VCR cells with the help of Lipofectamine 2000. The expressions of miR-15a and miR-16 mRNA were detected by RT-qPCR. The levels of bcl-2 and P-gp proteins were measured by Western blot. The inhibitory effects of VCR on growth of HCT8 and HCT8/VCR cells were detected by CCK8.</p><p><b>RESULTS</b>After transfection of the mimics, the expression of miR-15a in the blank control group, negative control group and miR-15a mimic group was 1.00, 0.87 ± 0.24, and 223.44 ± 59.07, respectively, and miR-15a was increased significantly (P < 0.001). The expression of miR-16 in the blank control group, negative control group and miR-16 mimic group was 1.00, 0.66 ± 0.19, and 107.32 ± 22.58, respectively, and miR-16 expression was increased significantly (P < 0.001). The Western blot assay showed that the relative expressions of bcl-2 protein in the blank control group, negative control group, miR-15a mimic group and miR-16 mimic group were 1.00, 0.97 ± 0.02, 0.51 ± 0.06, and 0.65 ± 0.03, respectively, and the expression of bcl-2 protein was decreased significantly (P < 0.05), however, the expressions of P-gp protein showed no significant difference. The CCK8 test showed that at 1, 5, 25 and 125 µg/ml concentration of VCR, the survival rates of HCT8/VCR cells were basically the same in the blank control group, negative control group, miR-15a mimic group and miR-16 mimic group, but the survival rate of HCT8/VCR cells was significantly decreased after transfection of mimics (P < 0.05). After transfection of the inhibitors, the expressions of both miR-15a and miR-16 were decreased significantly (P < 0.001). The Western blot showed that the expression of bcl-2 protein was increased (P < 0.05), while the expression of P-gp protein showed no significant difference. The CCK8 test showed that the survival rate of HCT8 cells which were transfected with inhibitors was significantly higher than that of the blank control group (P < 0.05).</p><p><b>CONCLUSIONS</b>miR-15a and miR-16 may reverse the drug resistance in human colon cancer cells. A possible mechanism is regulating the expression of bcl-2.</p>