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.

Plague pandemics and outbreaks have killed millions of people during the history of humankind. The disease, caused by the bacteria Yersinia pestis, is currently treated effectively with antibiotics. However, in the case of multidrug-resistant (MDR) bacteria, alternative treatments are required. Bacteriophage (phage) therapy has shown efficient antibacterial activity in various experimental animal models and in human patients infected with different MDR pathogens. Here, we evaluated the efficiency of фA1122 and PST phage therapy, alone or in combination with second-line antibiotics, using a well-established mouse model of pneumonic plague. Phage treatment significantly delayed mortality and limited bacterial proliferation in the lungs. However, the treatment did not prevent bacteremia, suggesting that phage efficiency may decrease in the circulation. Indeed, in vitro phage proliferation assays indicated that blood exerts inhibitory effects on lytic activity, which may be the major cause of treatment inefficiency. Combining phage therapy and second-line ceftriaxone treatment, which are individually insufficient, provided protection that led to the survival of all infected animals—a synergistic protective effect that represents a proof of concept for efficient combinatorial therapy in an emergency event of a plague outbreak involving MDR Y. pestis strains.

The generation of adaptive immunity by vaccination is usually a prolonged process that requires multiple dosing over several months. Hence, vaccines are administered for disease prevention a relatively long time prior to possible infection as opposed to post-exposure prophylaxis, which typically requires rapid intervention such as antibiotic therapy. The emergence of pathogens resistant to common antibiotic treatments has prompted the search for alternative therapeutic strategies. We previously demonstrated that vaccination of mice with the F1 capsular antigen ofYersinia pestiselicits specific and effective yet, unexpectedly, rapid anti-plague immunity. Here, we show by applying genetic and immunological approaches that the F1 antigen is targeted by peritoneal innate-like B1b cells that generate a prompt T-independent (TI) anti-F1 humoral response. The rapid F1-mediated defense response was diminished inXid(Btk^m) mice in which B1 cell numbers and activity are limited. Binding of fluorophore-labeled F1 to peritoneal B1b cells was detected as soon as 6 h post vaccination, emphasizing the high speed of this process. By assessing the ability to achieve rapid immunity with monomerized F1, we show that the natural polymeric structure of F1 is essential for (i) rapid association with peritoneal B1b cells, (ii) early induction of anti-F1 titers and (iii) rapid TI immunity in the mouse model of bubonic plague. These observations shed new light on the potential of novel as well as well-known protective antigens in generating rapid immunity and could be implemented in the rational design of future vaccines.

In a recent study, we demonstrated that vaccination with the polymeric F1 capsule antigen of the plague pathogen Yersinia pestis led to the rapid induction of a protective humoral immune response via the pivotal activation of innate-like B1b cells. Conversely, the monomeric version of F1 failed to promptly protect vaccinated animals in this model of the bubonic plague. In this study, we examined the ability of F1 to confer the rapid onset of protective immunity in the more challenging mouse model of the pneumonic plague. Vaccination with one dose of F1 adsorbed on aluminum hydroxide elicited effective protection against subsequent lethal intranasal exposure to a fully virulent Y. pestis strain within a week. Interestingly, the addition of the LcrV antigen shortened the time required for achieving such rapid protective immunity to 4–5 days after vaccination. As found previously, the polymeric structure of F1 was essential in affording the accelerated protective response observed by covaccination with LcrV. Finally, in a longevity study, a single vaccination with polymeric F1 induced a higher and more uniform humoral response than a similar vaccination with monomeric F1. However, in this setting, the dominant contribution of LcrV to long-lasting immunity against a lethal pulmonary challenge was reiterated.

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.

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.