Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (?30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.     

Exceptionally Potent and Broadly Cross-Reactive,Bispecific Multivalent HIV-1 Inhibitors Based on Single Human CD4 and Antibody Domains

Soluble forms of the human immunodeficiency virus type 1 (HIV-1) primary receptor CD4 (soluble CD4 [sCD4]) have been extensively characterized for a quarter of a century as promising HIV-1 inhibitors, but they have not been clinically successful. By combining a protein cavity-filling strategy and the power of library technology, we identified an engineered cavity-altered single-domain sCD4 (mD1.22) with a unique combination of excellent properties, including broad and potent neutralizing activity, high specificity, stability, solubility, and affinity for the HIV-1 envelope glycoprotein gp120, and small molecular size. To further improve its neutralizing potency and breadth, we generated bispecific multivalent fusion proteins of mD1.22 with another potent HIV-1 inhibitor, an antibody domain (m36.4) that targets the coreceptor-binding site on gp120. The fusion proteins neutralized all HIV-1 isolates tested, with potencies about 10-, 50-, and 200-fold higher than those of the broadly neutralizing antibody VRC01, the U.S. FDA-approved peptide inhibitor T20, and the clinically tested sCD4-Fc fusion protein CD4-Ig, respectively. In addition, they exhibited higher stability and specificity and a lower aggregation propensity than CD4-Ig. Therefore, mD1.22 and related fusion proteins could be useful for HIV-1 prevention and therapy, including eradication of the virus.     

Selection of non-competitive leptin antagonists using a random nanobody-based approach

The adipocyte-derived cytokine leptin acts as a metabolic switch, connecting the body's metabolism to high-energy consuming processes such as reproduction and immune responses. Accumulating evidence suggests that leptin plays a role in human pathologies, such as autoimmune diseases and cancer, thus providing a rationale for the development of leptin antagonists. In the present study, we generated and evaluated a panel of neutralizing nanobodies targeting the LR (leptin receptor). A nanobody comprises the variable domain of the naturally occurring single-chain antibodies found in members of the Camelidae family. We identified three classes of neutralizing nanobodies targeting different LR subdomains: i.e. the CRH2 (cytokine receptor homology 2), Ig-like and FNIII (fibronectin type?III) domains. Only nanobodies directed against the CRH2 domain inhibited leptin binding. We could show that a nanobody that targets the Ig-like domain potently interfered with leptin-dependent regulation of hypothalamic NPY (neuropeptide Y) expression. As a consequence, daily intraperitoneal injection increased body weight, body fat content, food intake, liver size and serum insulin levels. All of these characteristics resemble the phenotype of leptin and LR-deficient animals. The results of the present study support proposed models of the activated LR complex, and demonstrate that it is possible to block LR signalling without affecting ligand binding. These nanobodies form new tools to study the mechanisms of BBB (blood-brain barrier) leptin transport and the effect of LR inhibition in disease models.     

双特异性纳米抗体的研究进展及其应用

从纳米抗体的研究进展,双特异性纳米抗体在感染类疾病、肿瘤以及免疫系统疾病治疗领域的研究成果、研究热点及发展前景等方面综述了双特异性纳米抗体的研究进展并分析了未来可能的发展方向.首先比较了纳米抗体与全长单克隆抗体之间的差异并阐述了双特异性纳米抗体具备的独特优势;继而概括了双特异性纳米抗体的发展历程,并对新冠病毒的中和性抗...     

Biparatopic sybodies neutralize SARS‐CoV‐2 variants of concern and mitigate drug resistance

The ongoing COVID‐19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS‐CoV‐2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo‐EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri‐bispecific fusion constructs that exhibit up to 100‐ and 1,000‐fold increase in neutralization potency, respectively. Cryo‐EM of the sybody‐spike complex additionally reveals a novel up‐out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS‐CoV‐2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS‐CoV‐2 escape mutants.     

The Molecular Mechanism of Shiga Toxin Stx2e Neutralization by a Single-domain Antibody Targeting the Cell Receptor-binding Domain

Shiga toxin Stx2e is the major known agent that causes edema disease in newly weaned pigs. This severe disease is characterized by neurological disorders, hemorrhagic lesions, and frequent fatal outcomes. Stx2e consists of an enzymatically active A subunit and five B subunits that bind to a specific glycolipid receptor on host cells. It is evident that antibodies binding to the A subunit or the B subunits of Shiga toxin variants may have the capability to inhibit their cytotoxicity. Here, we report the discovery and characterization of a VHH single domain antibody (nanobody) isolated from a llama phage display library that confers potent neutralizing capacity against Stx2e toxin. We further present the crystal structure of the complex formed between the nanobody (NbStx2e1) and the Stx2e toxoid, determined at 2.8 Å resolution. Structural analysis revealed that for each B subunit of Stx2e, one NbStx2e1 is interacting in a head-to-head orientation and directly competing with the glycolipid receptor binding site on the surface of the B subunit. The neutralizing NbStx2e1 can in the future be used to prevent or treat edema disease.     

Expression Cloning of Camelid Nanobodies Specific for Xenopus Embryonic Antigens

Developmental biology relies heavily on the use of conventional antibodies, but their production and maintenance involves significant effort. Here we use an expression cloning approach to identify variable regions of llama single domain antibodies (known as nanobodies), which recognize specific embryonic antigens. A nanobody cDNA library was prepared from lymphocytes of a llama immunized with Xenopus embryo lysates. Pools of bacterially expressed cDNAs were sib-selected for the ability to produce specific staining patterns in gastrula embryos. Three different nanobodies were isolated: NbP1 and NbP3 stained yolk granules, while the reactivity of NbP7 was predominantly restricted to the cytoplasm and the cortex. The isolated nanobodies recognized specific protein bands in immunoblot analysis. A reverse proteomic approach identified NbP1 target antigen as EP45/Seryp, a serine protease inhibitor. Given the unique stability of nanobodies and the ease of their expression in diverse systems, we propose that nanobody cDNA libraries represent a promising resource for molecular markers for developmental biology.     

The development of CD4 binding site antibodies during HIV-1 infection

Broadly neutralizing antibodies to the CD4 binding site (CD4bs) of gp120 are generated by some HIV-1-infected individuals, but little is known about the prevalence and evolution of this antibody response during the course of HIV-1 infection. We analyzed the sera of 113 HIV-1 seroconverters from three cohorts for binding to a panel of gp120 core proteins and their corresponding CD4bs knockout mutants. Among sera collected between 99 and 258 weeks post-HIV-1 infection, 88% contained antibodies to the CD4bs and 47% contained antibodies to resurfaced stabilized core (RSC) probes that react preferentially with broadly neutralizing CD4bs antibodies (BNCD4), such as monoclonal antibodies (MAbs) VRC01 and VRC-CH31. Analysis of longitudinal serum samples from a subset of 18 subjects revealed that CD4bs antibodies to gp120 arose within the first 4 to 16 weeks of infection, while the development of RSC-reactive antibodies was more varied, occurring between 10 and 152 weeks post-HIV-1 infection. Despite the presence of these antibodies, serum neutralization mediated by RSC-reactive antibodies was detected in sera from only a few donors infected for more than 3 years. Thus, CD4bs antibodies that bind a VRC01-like epitope are often induced during HIV-1 infection, but the level and potency required to mediate serum neutralization may take years to develop. An improved understanding of the immunological factors associated with the development and maturation of neutralizing CD4bs antibodies during HIV-1 infection may provide insights into the requirements for eliciting this response by vaccination.     

Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin

A less than adequate therapeutic plan for the treatment of anthrax in the 2001 bioterrorism attacks has highlighted the importance of developing alternative or complementary therapeutic approaches for biothreat agents. In these regards passive immunization possesses several important advantages over active vaccination and the use of antibiotics, as it can provide immediate protection against Bacillus anthracis. Herein, we report the selection and characterization of several human monoclonal neutralizing antibodies against the toxin of B. anthracis from a phage displayed human scFv library. In total 15 clones were selected with distinct sequences and high specificity to protective antigen and thus were the subject of a series of both biophysical and cell-based cytotoxicity assays. From this panel of antibodies a set of neutralizing antibodies were identified, of which clone A8 recognizes the lethal (and/or edema) factor binding domain, and clones F1, G11, and G12 recognize the cellular receptor binding domain found within the protective antigen. It was noted that all clones distinguish a conformational epitope existing on the protective antigen; this steric relationship was uncovered using a sequential epitope mapping approach. For each neutralizing antibody, the kinetic constants were determined by surface plasmon resonance, while the potency of protection was established using a two-tier macrophage cytotoxicity assay. Among the neutralizing antibodies identified, clone F1 possessed the highest affinity to protective antigen, and provided superior protection from lethal toxin in the cell cytotoxicity assay. The data presented provide the ever-growing arsenal of immunological and functional analysis of monoclonal antibodies to the exotoxins of anthrax. In addition it grants new candidates for the prophylaxis and therapeutic treatment against this toxin.     

抗人类免疫缺陷病毒1型感染的双特异性抗体的构建及其功能研究

靶向人类免疫缺陷病毒（human immunodeficiency virus，HIV）流行毒株的广谱中和抗体（broadly neutralizing antibody, bNAb）单一疗法最终会导致机体出现病毒逃逸突变，然而基于广谱中和抗体开发的双特异性或多特异性抗体则表现出较好的中和效力和广谱性。根据已公布的单链可变区基因抗体序列，经密码子优化后合成一种由单基因编码的双特异性抗体iMab-PGT151，经双酶切和测序对重组质粒进行了验证。酶联免疫吸附试验检测双特异性抗体的结合特异性，检测U87细胞裂解液中的荧光素酶活性以定量分析双特异性抗体对HIV-1假病毒的中和作用；间接免疫荧光染色法检测双特异性抗体iMab-PGT151对人喉癌上皮细胞的反应性；酶联免疫吸附试验检测该抗体对心磷脂的结合能力，验证其自体反应性。结果显示，构建的双特异抗体iMab-PGT151能够成功表达，可分别结合亲本抗体的各个配体，具有双特异性，能100%中和20株假病毒，IC50值为0.084 μg/mL。与亲本抗体相比，该抗体具有更强的中和效力和广谱度，无自体反应性，具有临床适用性。所构建的双特异性抗体iMab-PGT151将可能成为预防和治疗HIV-1感染的有效候选药物之一。     

Structural mimicry of CD4 by a cross-reactive HIV-1 neutralizing antibody with CDR-H2 and H3 containing unique motifs

Human immunodeficiency virus (HIV) entry into cells is initiated by the binding of its envelope glycoprotein (Env) gp120 to receptor CD4. Antibodies that bind to epitopes overlapping the CD4-binding site (CD4bs) on gp120 can prevent HIV entry by competing with cell-associated CD4; their ability to outcompete CD4 is a major determinant of their neutralizing potency and is proportional to their avidity. The breadth of neutralization and the likelihood of the emergence of antibody-resistant virus are critically dependent on the structure of their epitopes. Because CD4bs is highly conserved, it is reasonable to hypothesize that antibodies closely mimicking CD4 could exhibit relatively broad cross-reactivity and a high probability of preventing the emergence of resistant viruses. Previously, in a search for antibodies that mimic CD4 or the co-receptor, we identified and characterized a broadly cross-reactive HIV-neutralizing CD4bs human monoclonal antibody (hmAb), m18. Here, we describe the crystal structure of Fab m18 at 2.03 A resolution, which reveals unique conformations of heavy chain complementarity-determining regions (CDRs) 2 and 3 (H2 and H3). H2 is highly bulged and lacks cross-linking interstrand hydrogen bonds observed in all four canonical structures. H3 is 17.5 A long and rigid, forming an extended beta-sheet decorated with an alpha-turn motif bearing a phenylalanine-isoleucine fork at the apex. It shows striking similarity to the Ig CDR2-like C'C' region of the CD4 first domain D1 that dominates the binding of CD4 to gp120. Docking simulations suggest significant similarity between the m18 epitope and the CD4bs on gp120. Fab m18 does not enhance binding of CD4-induced (CD4i) antibodies, nor does it induce CD4-independent fusion mediated by the HIV Env. Thus, vaccine immunogens based on the m18 epitope structure are unlikely to elicit antibodies that could enhance infection. The structure can also serve as a basis for the design of novel, highly efficient inhibitors of HIV entry.     

Applications of nanobodies in plant science and biotechnology

Key message

Present review summarizes the current applications of nanobodies in plant science and biotechnology, including plant expression of nanobodies, plant biotechnological applications, nanobody-based immunodetection, and nanobody-mediated resistance against plant pathogens.

Abstract

Nanobodies (Nbs) are variable domains of heavy chain-only antibodies (HCAbs) isolated from camelids. In spite of their single domain structure, nanobodies display many unique features, such as small size, high stability, and cryptic epitopes accessibility, which make them ideal for sophisticated applications in plants and animals. In this review, we summarize the current applications of nanobodies in plant science and biotechnology, focusing on nanobody expression in plants, plant biotechnological applications, determination of plant toxins and pathogens, and nanobody-mediated resistance against plant pathogens. Prospects and challenges of nanobody applications in plants are also discussed.

     

Biochemically defined HIV-1 envelope glycoprotein variant immunogens display differential binding and neutralizing specificities to the CD4-binding site

HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.     

不同给药途径的治疗性纳米抗体研究进展

骆驼科及鲨鱼科动物血清中天然存在的纳米抗体具有不同于传统单克隆抗体的独特结构和分子量,这为抗体药物开发提供了全新的思路。纳米抗体较小的分子量和优异的稳定性使其在给药方面具有更大的灵活性,可以在一定程度上克服传统单克隆抗体在给药途径方面存在的局限性。同时,较小的分子量使纳米抗体具有双重药代动力学特征,既有优异的组织渗透性,又表现出快速的血液清除。重点介绍纳米抗体的药物代谢动力学特征和进一步改善药代动力学的方法,综述不同给药途径的纳米抗体药物研究进展,对其治疗特定疾病的可行性、安全性以及治疗效果进行分析,以期为纳米抗体药物研发中给药途径的选择提供参考。     

针对炭疽保护性抗原不同结构域的中和性单克隆抗体的筛选和鉴定

炭疽保护性抗原（PA）是炭疽毒素的重要组分,同时也是现有炭疽疫苗的主要有效成分,在炭疽杆菌的致病与免疫中发挥关键作用。以重组PA为免疫原,采用B淋巴细胞杂交瘤技术,结合炭疽毒素敏感细胞的毒性中和试验,大量筛选抗PA单克隆抗体,获得了9株炭疽毒素中和性单抗。进一步分析表明这些单抗以IgG1亚类为主,分别识别PA 3个结构域的4个不同中和表位区。针对结构域2的4株单抗识别同一表位区,其中3株单抗的中和活性强于抗PA多抗;针对结构域4的4株单抗识别两个不同表位区;另有1株单抗识别位于结构域3的表位。实验结果提示PA具有多个中和表位,分别位于其不同结构域,其中结构域2、4包含主要中和表位。实验中获得的针对不同表位的中和性单抗为深入研究PA的免疫保护机理提供了工具,也为研制针对炭疽毒素的被动免疫制剂和治疗药物打下基础。     

Methods for analysis of biologically functional antibodies to the HIV-1 gp120 principal neutralizing domain.

The humoral response to HIV-1 infection has been demonstrated by a variety of immunoassays utilizing viral proteins. While several assays detect HIV-1 infection with high sensitivity and great specificity, little progress has been made to develop immunoassays correlative with disease progression and viral transmission. Antibodies toward the V3 domain of HIV-1 envelope can prevent virus infection and block virus-mediated cell fusion in vitro. Such properties may be critical to the course of the disease. Furthermore, understanding the role of neutralizing antibodies against HIV-1 during infection in humans and generating biologically relevant neutralizing antibodies are paramount to developing an efficacious AIDS vaccine. In this study we explored peptide binding and neutralization assays and their relation to predicting disease progression and viral transmission. Biologically relevant polyclonal and monoclonal neutralizing antibodies that were derived from natural HIV-1 infection of humans, experimental infections of chimpanzees, and viral envelope protein peptide immunizations were characterized. Comparison of V3-specific monoclonal antibodies by antigen-limited ELISA and a quantitative HIV-1 neutralization assay demonstrated a less than optimal predictive relationship between binding and neutralization potency. On the other hand, polyclonal sera from goats immunized with V3-specific peptides derived from three different HIV-1 strains, as well as sera from other HIV-1-infected individuals demonstrated correlation between binding affinity and neutralization.     

Enfuvirtide resistance mutations: impact on human immunodeficiency virus envelope function, entry inhibitor sensitivity, and virus neutralization

Enfuvirtide (ENF/T-20/Fuzeon), the first human immunodeficiency virus (HIV) entry inhibitor to be licensed, targets a structural intermediate of the entry process. ENF binds the HR1 domain in gp41 after Env has bound CD4, preventing conformational changes needed for membrane fusion. Mutations in HR1 that confer ENF resistance can arise following ENF therapy. ENF resistance mutations were introduced into an R5- and X4-tropic Env to examine their impact on fusion, infection, and sensitivity to different classes of entry inhibitors and neutralizing antibodies. HR1 mutations could reduce infection and fusion efficiency and also delay fusion kinetics, likely accounting for their negative impact on viral fitness. HR1 mutations had minimal effect on virus sensitivity to other classes of entry inhibitors, including those targeting CD4 binding (BMS-806 and a CD4-specific monoclonal antibody [MAb]), coreceptor binding (CXCR4 inhibitor AMD3100 and CCR5 inhibitor TAK-779), or fusion (T-1249), indicating that ENF-resistant viruses can remain sensitive to other entry inhibitors in vivo. Some HR1 mutations conferred increased sensitivity to a subset of neutralizing MAbs that likely target fusion intermediates or with epitopes preferentially exposed following receptor interactions (17b, 48D, 2F5, 4E10, and IgGb12), as well as sera from some HIV-positive individuals. Mechanistically, enhanced neutralization correlated with reduced fusion kinetics, indicating that, in addition to steric constraints, kinetics may also limit virus neutralization by some antibodies. Therefore, escape from ENF comes at a cost to viral fitness and may confer enhanced sensitivity to humoral immunity due to prolonged exposure of epitopes that are not readily accessible in the native Env trimer. Resistance to other entry inhibitors was not observed.     

Neutralization of SARS‐CoV‐2 by highly potent,hyperthermostable, and mutation‐tolerant nanobodies

Monoclonal anti‐SARS‐CoV‐2 immunoglobulins represent a treatment option for COVID‐19. However, their production in mammalian cells is not scalable to meet the global demand. Single‐domain (VHH) antibodies (also called nanobodies) provide an alternative suitable for microbial production. Using alpaca immune libraries against the receptor‐binding domain (RBD) of the SARS‐CoV‐2 Spike protein, we isolated 45 infection‐blocking VHH antibodies. These include nanobodies that can withstand 95°C. The most effective VHH antibody neutralizes SARS‐CoV‐2 at 17–50 pM concentration (0.2–0.7 µg per liter), binds the open and closed states of the Spike, and shows a tight RBD interaction in the X‐ray and cryo‐EM structures. The best VHH trimers neutralize even at 40 ng per liter. We constructed nanobody tandems and identified nanobody monomers that tolerate the K417N/T, E484K, N501Y, and L452R immune‐escape mutations found in the Alpha, Beta, Gamma, Epsilon, Iota, and Delta/Kappa lineages. We also demonstrate neutralization of the Beta strain at low‐picomolar VHH concentrations. We further discovered VHH antibodies that enforce native folding of the RBD in the E. coli cytosol, where its folding normally fails. Such “fold‐promoting” nanobodies may allow for simplified production of vaccines and their adaptation to viral escape‐mutations.     

Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU

The epitope specificities and functional activities of monoclonal antibodies (MAbs) specific for the murine leukemia virus (MuLV) SU envelope protein subunit were determined. Neutralizing antibodies were directed towards two distinct sites in MuLV SU: one overlapping the major receptor-binding pocket in the N-terminal domain and the other involving a region that includes the most C-terminal disulfide-bonded loop. Two other groups of MAbs, reactive with distinct sites in the N-terminal domain or in the proline-rich region (PRR), did not neutralize MuLV infectivity. Only the neutralizing MAbs specific for the receptor-binding pocket were able to block binding of purified SU and MuLV virions to cells expressing the ecotropic MuLV receptor, mCAT-1. Whereas the neutralizing MAbs specific for the C-terminal domain did not interfere with the SU-mCAT-1 interaction, they efficiently inhibited cell-to-cell fusion mediated by MuLV Env, indicating that they interfered with a postattachment event necessary for fusion. The C-terminal domain MAbs displayed the highest neutralization titers and binding activities. However, the nonneutralizing PRR-specific MAbs bound to intact virions with affinities similar to those of the neutralizing receptor-binding pocket-specific MAbs, indicating that epitope exposure, while necessary, is not sufficient for viral neutralization by MAbs. These results identify two separate neutralization domains in MuLV SU and suggest a role for the C-terminal domain in a postattachment step necessary for viral fusion.     

NeutrobodyPlex—monitoring SARS‐CoV‐2 neutralizing immune responses using nanobodies

In light of the COVID‐19 pandemic, there is an ongoing need for diagnostic tools to monitor the immune status of large patient cohorts and the effectiveness of vaccination campaigns. Here, we present 11 unique nanobodies (Nbs) specific for the SARS‐CoV‐2 spike receptor‐binding domain (RBD), of which 8 Nbs potently inhibit the interaction of RBD with angiotensin‐converting enzyme 2 (ACE2) as the major viral docking site. Following detailed epitope mapping and structural analysis, we select two inhibitory Nbs, one of which binds an epitope inside and one of which binds an epitope outside the RBD:ACE2 interface. Based on these, we generate a biparatopic nanobody (bipNb) with viral neutralization efficacy in the picomolar range. Using bipNb as a surrogate, we establish a competitive multiplex binding assay (“NeutrobodyPlex”) for detailed analysis of the presence and performance of neutralizing RBD‐binding antibodies in serum of convalescent or vaccinated patients. We demonstrate that NeutrobodyPlex enables high‐throughput screening and detailed analysis of neutralizing immune responses in infected or vaccinated individuals, to monitor immune status or to guide vaccine design.