Search Results (6)

Background: Rift Valley fever virus (RVFV) is a zoonotic virus that poses a significant threat to both livestock and human health and has caused outbreaks in endemic regions. In humans, most patients experience a febrile illness; however, in some patients, RVF disease may result in hemorrhagic fever, retinitis, or encephalitis. While several veterinary vaccines are being utilized in endemic countries, currently, there are no licensed RVF vaccines or therapeutics for human use. Neutralizing antibodies specifically targeting vulnerable pathogen epitopes are promising candidates for prophylactic and therapeutic interventions. In the case of RVFV, the surface glycoproteins Gc and Gn, which harbor neutralizing epitopes, represent the primary targets for vaccine and neutralizing antibody development. Methods: We report the implementation of advanced 10x Genomics technology, enabling high-throughput single-cell analysis for the identification of rare and potent antibodies against RVFV. Following the immunization of mice with live attenuated rMP-12-GFP virus and successive Gc/Gn boosts, memory B cell populations (both general and antigen-specific) were sorted from splenocytes by flow cytometry. Deep sequencing of the antibody repertoire at a single-cell resolution, together with bioinformatic analyses, was applied for BCR pair selection based on their abundance and specificity. Results: Twenty-three recombinant monoclonal antibodies (mAbs) were selected and expressed, and their antigen-binding capacities were characterized. About half of them demonstrated specific binding to their cognate antigen with relatively high binding affinities. Conclusions: These antibodies could be used for the future development of efficacious therapeutics, as well as for studying virus-neutralizing mechanisms. The current study, in which the single-cell sequencing approach was implemented for the development of antibodies targeting the RVFV surface proteins Gc and Gn, demonstrates the effective applicability of this technique for antibody discovery purposes. Full article

SARS-CoV-2 surface spike protein mediates the viral entry into the host cell and represents the primary immunological target of COVID-19 vaccines as well as post-exposure immunotherapy. Establishment of the highly immunogenic B-cell epitope profile of SARS-CoV-2 proteins in general, and that of the spike protein in particular, may contribute to the development of sensitive diagnostic tools and identification of vaccine` candidate targets. In the current study, the anti-viral antibody response in transgenic K18-hACE-2 mice was examined by implementing an immunodominant epitope mapping approach of the SARS-CoV-2 spike. Serum samples for probing an epitope array covering the entire spike protein were collected from mice following infection with the original SARS-CoV-2 strain as well as the B.1.1.7 Alpha and B.1.351 Beta genetic variants of concern. The analysis resulted in distinction of six linear epitopes common to the humoral response against all virus variants inspected at a frequency of more than 20% of the serum samples. Finally, the universality of the response was probed by cross-protective in vitro experiments using plaque-reducing neutralization tests. The data presented here has important implications for prediction of the efficacy of immune countermeasures against emerging SARS-CoV-2 variants. Full article

The use of passively-administered neutralizing antibodies is a promising approach for the prevention and treatment of SARS-CoV-2 infection. Antibody-mediated protection may involve immune system recruitment through Fc-dependent activation of effector cells and the complement system. However, the role of Fc-mediated functions in the efficacious in-vivo neutralization of SARS-CoV-2 is not yet clear, and it is of high importance to delineate the role this process plays in antibody-mediated protection. Toward this aim, we have chosen two highly potent SARS-CoV-2 neutralizing human monoclonal antibodies, MD65 and BLN1 that target distinct domains of the spike (RBD and NTD, respectively). The Fc of these antibodies was engineered to include the triple mutation N297G/S298G/T299A that eliminates glycosylation and the binding to FcγR and to the complement system activator C1q. As expected, the virus neutralization activity (in-vitro) of the engineered antibodies was retained. To study the role of Fc-mediated functions, the protective activity of these antibodies was tested against lethal SARS-CoV-2 infection of K18-hACE2 transgenic mice, when treatment was initiated either before or two days post-exposure. Antibody treatment with both Fc-variants similarly rescued the mice from death reduced viral load and prevented signs of morbidity. Taken together, this work provides important insight regarding the contribution of Fc-effector functions in MD65 and BLN1 antibody-mediated protection, which should aid in the future design of effective antibody-based therapies. Full article

Monoclonal antibodies represent an important avenue for COVID-19 therapy and are routinely used for rapid and accessible diagnosis of SARS-CoV-2 infection. The recent emergence of SARS-CoV-2 genetic variants emphasized the need to enlarge the repertoire of antibodies that target diverse epitopes, the combination of which may improve immune-diagnostics, augment the efficiency of the immunotherapy and prevent selection of escape-mutants. Antigen-specific controlled immunization of experimental animals may elicit antibody repertoires that significantly differ from those generated in the context of the immune response mounted in the course of disease. Accordingly, rabbits were immunized by several recombinant antigens representing distinct domains of the viral spike protein and monoclonal antibodies were isolated from single cells obtained by cell sorting. Characterization of a panel of successfully isolated anti-receptor binding domain (RBD) and anti-N-terminal domain (NTD) antibodies demonstrated that they exhibit high specificity and affinity profiles. Anti-RBD antibodies revealing significant neutralizing potency against SARS-CoV-2 in vitro were found to target at least three distinct epitopes. Epitope mapping established that two of these antibodies recognized a novel epitope located on the surface of the RBD. We suggest that the antibodies isolated in this study are useful for designing SARS-CoV-2 diagnosis and therapy approaches. Full article

Abrin toxin is a type 2 ribosome inactivating glycoprotein isolated from the seeds of Abrus precatorius (jequirity pea). Owing to its high toxicity, relative ease of purification and accessibility, it is considered a biological threat agent. To date, there is no effective post-exposure treatment for abrin poisoning and passive immunization remains the most effective therapy. However, the effectiveness of anti-abrin monoclonal antibodies for post-exposure therapy following abrin intoxication has not been demonstrated. The aim of this study was to isolate high affinity anti-abrin antibodies that possess potent toxin-neutralization capabilities. An immune scFv phage-display library was constructed from an abrin-immunized rabbit and a panel of antibodies (six directed against the A subunit of abrin and four against the B subunit) was isolated and expressed as scFv-Fc antibodies. By pair-wise analysis, we found that these antibodies target five distinct epitopes on the surface of abrin and that antibodies against all these sites can bind the toxin simultaneously. Several of these antibodies (namely, RB9, RB10, RB28 and RB30) conferred high protection against pulmonary intoxication of mice, when administered six hours post exposure to a lethal dose of abrin. The data presented in this study demonstrate for the first time the efficacy of monoclonal antibodies in treatment of mice after pulmonary intoxication with abrin and promote the use of these antibodies, one or several, for post-exposure treatment of abrin intoxication. Full article

Ricin, derived from the castor bean plant Ricinus communis, is one of the most potent and lethal toxins known, against which there is no available antidote. To date, the use of neutralizing antibodies is the most promising post-exposure treatment for ricin intoxication. The aim of this study was to isolate high affinity anti-ricin antibodies that possess potent toxin-neutralization capabilities. Two non-human primates were immunized with either a ricin-holotoxin- or subunit-based vaccine, to ensure the elicitation of diverse high affinity antibodies. By using a comprehensive set of primers, immune scFv phage-displayed libraries were constructed and panned. A panel of 10 antibodies (five directed against the A subunit of ricin and five against the B subunit) was isolated and reformatted into a full-length chimeric IgG. All of these antibodies were found to neutralize ricin in vitro, and several conferred full protection to ricin-intoxicated mice when given six hours after exposure. Six antibodies were found to possess exceptionally high affinity toward the toxin, with K_D values below pM (k_off < 1 × 10⁻⁷ s⁻¹) that were well correlated with their ability to neutralize ricin. These antibodies, alone or in combination, could be used for the development of a highly-effective therapeutic preparation for post-exposure treatment of ricin intoxication. Full article