Dromedary camel nanobodies broadly neutralize SARS-CoV-2 variants

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is a trimer of S1/S2 heterodimers with three receptor-binding domains (RBDs) at the S1 subunit for human angiotensin-converting enzyme 2 (hACE2). Due to their small size, nanobodies can recognize protein cavities that are not accessible to conventional antibodies. To isolate high-affinity nanobodies, large libraries with great diversity are highly desirable. Dromedary camels (Camelus dromedarius) are natural reservoirs of coronaviruses like Middle East respiratory syndrome CoV (MERS-CoV) that are transmitted to humans. Here, we built large dromedary camel V_HH phage libraries to isolate nanobodies that broadly neutralize SARS-CoV-2 variants. We isolated two V_HH nanobodies, NCI-CoV-7A3 (7A3) and NCI-CoV-8A2 (8A2), which have a high affinity for the RBD via targeting nonoverlapping epitopes and show broad neutralization activity against SARS-CoV-2 and its emerging variants of concern. Cryoelectron microscopy (cryo-EM) complex structures revealed that 8A2 binds the RBD in its up mode with a long CDR3 loop directly involved in the ACE2 binding residues and that 7A3 targets a deeply buried region that uniquely extends from the S1 subunit to the apex of the S2 subunit regardless of the conformational state of the RBD. At a dose of ≥5 mg/kg, 7A3 efficiently protected transgenic mice expressing hACE2 from the lethal challenge of variants B.1.351 or B.1.617.2, suggesting its therapeutic use against COVID-19 variants. The dromedary camel V_HH phage libraries could be helpful as a unique platform ready for quickly isolating potent nanobodies against future emerging viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of COVID-19 (1, 2) that enters human cells by binding its envelope anchored type I fusion protein (spike) to angiotensin-converting enzyme 2 (ACE2) (3, 4). The SARS-CoV-2 spike is a trimer of S1/S2 heterodimers with three ACE2 receptor-binding domains (RBDs) attached to the distal end of the spike via a hinge region that allows conformational flexibility (4). In the all-down conformation, the RBDs are packed with their long axes contained in a plane perpendicular to the axis of symmetry of the trimer. Transition to the roughly perpendicular up conformation exposes the receptor-binding motif (RBM), located at the distal end of the RBD, which is sterically occluded in the down state. Numerous neutralizing antibodies targeting the spike, particularly its RBD, have been developed to treat COVID-19 using common strategies such as single B cell cloning, animal immunization, and phage display (5–9). Most vaccines, including those that are messenger RNA based, are designed to induce immunity against the spike or RBD (10–12). However, emerging SARS-CoV-2 variants such as D614G, B.1.1.7 (Alpha, United Kingdom), B.1.351 (Beta, South Africa), and P.1 (Gamma, Brazil) have exhibited increased resistance to neutralization by monoclonal antibodies or postvaccination sera elicited by the COVID-19 vaccines (13, 14). Monoclonal antibodies with Emergency Use Authorization for COVID-19 treatment partially (Casirivimab) or completely (Bamlanivimab) failed to inhibit the B.1.351 and P.1 variants. Similarly, these variants were less effectively inhibited by convalescent plasma and sera from individuals vaccinated with a COVID-19 vaccine (BNT162b2) (13). The B.1.617.2 (Delta, India) variant became the prevailing strain in many countries (15). Highly effective and broadly neutralizing antibody therapy is urgently demanded for COVID-19 patients.Due to their small size and unique conformations, camelid V_HH single-domain antibodies (also known as nanobodies) can recognize protein cavities that are not accessible to conventional antibodies (16). To isolate high-affinity nanobodies without a need for further affinity maturation, it is highly desirable to construct large nanobody libraries with great diversity. Dromedary camels have been found as potential natural reservoirs of Middle East respiratory syndrome CoV (MERS-CoV) (17). We speculated that dromedary camels would be an ideal source of neutralizing nanobodies against coronaviruses. In the present study, we built large camel V_HH single-domain antibody phage libraries with a diversity of over 10¹¹ from six dromedary camels (Camelus dromedarius), three males and three females, with ages ranging from 3 mo to 20 y. We used both the SARS-CoV-2 RBD and the stabilized spike ectodomain trimer protein as baits to conduct phage panning for nanobody screening. Among all the binders, we found NCI-CoV-7A3 (7A3), NCI-CoV-1B5 (1B5), NCI-CoV-8A2 (8A2), and NCI-CoV-2F7 (2F7) to be potent ACE2 blockers. In addition, these dromedary camel nanobodies displayed potent neutralization activity against the B.1.351 and B.1.1.7 variants and the original strain (Wuhan-Hu-1). The cryoelectron microscopy (cryo-EM) structure of the spike trimer protein complex with these V_HH nanobodies revealed two distinct nonoverlapping epitopes for neutralizing SARS-CoV-2. In particular, 7A3 recognizes a unique and deeply buried region that extends to the apex of the S2 subunit of the spike. Combined treatment with 7A3 and 8A2 shows more potent protection against various variants in culture and mice infected with the B.1.351 variant. Interestingly, 7A3 alone retains its neutralization activity against the lethal challenge of the B.1.617.2 variant in mice.     

Use of a digital hearing aid with directional microphones in school-aged children

The efficacy of a digital hearing aid with a directional microphone was examined in a school-aged population. Twenty children (9 with a mild-to-moderately-severe hearing loss and 11 with a moderate-to-severe hearing loss) between 7 1/2 and 13 2/3 years of age wore the study hearing aids binaurally for 30 days prior to the evaluation. The testing protocol included speech recognition tests using the CID W-22 word lists presented at 72 dB SPL, 65 dB SPL, and 52 dB SPL (at 0 degrees azimuth) in the presence of a 65 dB SPL party noise (180 degrees azimuth). Subjective rating of hearing aid efficacy in the classroom was examined using the Listening Inventory For Education (LIFE) questionnaire. Parental impression on hearing aid efficacy was also collected at the end of the study. The results showed improved speech recognition in noise with the digital directional hearing aid at all presentation levels. Preference for the digital directional hearing aids over the subjects' own omnidirectional analog hearing aids was also seen on the LIFE questionnaire and parental impression. The degree of hearing loss did not seem to have affected the benefits offered by the digital directional hearing aids. These results were compared to results from other studies on the use of directional microphones in hearing aids.     

Aortic dilation, dissection, and rupture in patients with Turner syndrome

We report two patients with Turner syndrome who had aortic dissection and rupture, one with prior repair of coarctation. We also note the high incidence (8.8%) of unrecognized aortic root dilation in a group of 57 patients with Turner syndrome whom we prospectively evaluated by echocardiography. Our analysis and review of previously reported cases suggests that multiple risk factors may exist for aortic dissection, including coarctation, bicuspid aortic valve, and systemic hypertension, but that these need not be present. Aortic root dilation may be an additional finding that suggests the patient with Turner syndrome is also at risk. When it is present, magnetic resonance imaging visualizes the entire aorta and allows quantification of the site and degree of dilation. In patients with dissection, the aorta often exhibits pathologic evidence of cystic medial necrosis similar to the finding in patients with Marfan syndrome. Therapeutic methods to decrease risk, such as those directed toward prevention of bacterial endocarditis, blood pressure control, and perhaps prophylactic beta blockade or surgical reconstruction, may need to be considered. Patients with Turner syndrome, their families, and the physicians who care for them should be aware of the significance of unexplained chest pain, dyspnea, or hypotension as potential manifestations of aortic dissection or rupture.     

Successful treatment of pediatric plasmacytoid dendritic cell tumors with a contemporary regimen for acute lymphoblastic leukemia

Dendritic cell leukemia (DCL) or hematodermic tumor is an uncommon subtype of acute leukemia. In contrast to adult cases, children tend to have a less aggressive course. The diagnosis of DCL should be considered when its characteristic morphologic features are present and leukemic cells co‐express CD4 and CD56. Cases of DCL among pediatric patients have been reported to respond to therapeutic regimens for acute lymphoblastic leukemia, but details regarding the specifics of therapy are lacking. Pediatr Blood Cancer 2013; 60: E38–E41. © 2013 Wiley Periodicals, Inc.     

Aqueous amino acid levels in Fuchs' corneal dystrophy

Eight patients with Fuchs' corneal dystrophy and 11 matched senile cataract patients were examined at the time of surgery for aqueous humor amino acid levels. The cataract patients had amino acid levels that did not differ significantly from control values. The anterior chamber concentrations of threonine, glutamine, and arginine were found to be significantly increased in the patients with Fuchs' corneal dystrophy. Asparagine, phosphoserine, and phosphoethanolamine were found to be significantly decreased in patients with Fuchs' dystrophy.     

Photohemolysis of human erythrocytes induced by aluminum phthalocyanine tetrasulfonate

The phthalocyanines are a new class of photosensitizers with promising properties for use in photodynamic therapy of cancer [E. Ben-Hur and I. Rosenthal (1985) Int. J. Radiat. Biol., 47, 145–147]. The ability of aluminum phthalocyanine tetrasulfonate (AlPCS) to cause membrane damage upon light exposure was tested using photohemolysis of human erythrocytes as an endpoint. AlPCS was found to be quite efficient in causing red blood cell lysis. Photohemolysis dependency on the light fluence had a characteristic sigmoidal shape. The light fluence required for 50% hemolysis was inversely proportional to AlPCS concentration. Neither singlet oxygen nor hydroxyl radicals appear to be involved in the photohemolysis induced by AlPCS. This is inferred from the observation that exposure of erythrocytes in the presence of D₂O or glycerol did not affect the light fluence response curve. These data suggest that photosensitization by AlPCS can cause membrane damage and that this damage may be responsible for cell killing.     

Antimicrobial comparison of a new multi-purpose disinfecting solution to a 3% hydrogen peroxide system.

PURPOSE: We compared the antimicrobial activity of a new multi-purpose disinfection solution (OPTI-FREE EXPRESS with ALDOX) to a 3% hydrogen peroxide disinfecting system. The antimicrobial ingredients in the new solution are polyquaternium-1 and myristamidopropyl dimethylamine. METHODS: The solutions were tested for antimicrobial activity against Staphylococcus sp.., Pseudomonas aeruginosa, Serratia marcescens, Candida albicans, Fusarium solani, and two Acanthamoeba species and were evaluated according to the primary criteria of the FDA and ISO contact lens disinfection procedures. RESULTS AND CONCLUSIONS: OPTI-FREE EXPRESS with ALDOX (EXPRESS MPDS) Multi-Purpose Disinfecting Solution provided a multiple spectrum of antimicrobial activity and met the FDA and ISO primary Stand Alone Test criteria for disinfection of contact lenses. EXPRESS MPDS showed disinfection activity in the range of a 3% hydrogen peroxide system. Unlike the 3% hydrogen peroxide system, it retarded contamination during storage.